A microfluidic DNA library preparation platform for next-generation sequencing

Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR

February 13, 2022 Lieven Leave a comment

Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations.

MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase https://biodas.org/ DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.

Sensory analysis of hepatitis B virus DNA for medicinal clinical diagnostics based on molybdenum doped ZnO nanowires field effect transistor biosensor; a comparative study to PCR test results

In this paper, a bio-sensing setup for investigating hepatitis B virus deoxyribonucleic acid (HBV DNA) diagnosis including rapid testing and field effect transistor (FET) in label free assay is proposed. The FET biosensor was fabricated by molybdenum doped ZnO nanowires (NWs) in easy method and cost-free approach. The materialized NWs were synthesized by physical vapor deposition (PVD) growth mechanism.

The molybdenum dopant could bring about vacancy sites for DNA adsorption and electric charge transfer. The capability of the fabricated biosensor was evaluated by investigating the PCR-verified samples known as True Positive (TP), True Negative (TN), False Positive (FP) and False Negative (FN). The FET biosensor could materialize the clinical tests on samples TP, TN, FP and FN and could distinguish the clinical samples from each other. The designed biosensor showed more precision than the PCR-outcomes by exhibiting more sensitivity on labeled samples known as FN.

This research has analytical and comparative study on fabricated biosensor performance. The achieved results show that the biosensor had significant response to samples which have not been carefully detected by PCR test. The fabricated biosensor showed high accuracy, precision, sensitivity, specificity and reproducibility for differentiating (TP), (TN), (FP) and (FN) samples from healthy and normal sample. The response sensitivity was calculated and biosensor showed a detection limit (LOD) of 1 pM. The biosensor demonstrated high response and satisfied signal in the concentration ranges from 1 pM to 10 μM.

ACE2-based capacitance sensor for rapid native SARS-CoV-2 detection in biological fluids and its correlation with real-time PCR

The spread of the SARS-CoV-2 and its increasing threat to human health worldwide have necessitated the development of new technological tools to combat the virus. Particular emphasis is given to the development of diagnostic methods that monitor the spread of the virus rapidly and effectively.
In this study, we report the development and testing of an antibody-free biosensor, based on the immobilization of ACE2 protein on the surface of gold interdigitated electrode. When the sensor was used in laboratory conditions for targeting the virus’ structural spike protein, it showed a limit of detection [LOD] of 750 pg/μL/mm². Thereafter, the response of the sensor to swab and saliva samples from hospitalized patients was examined.
The virus presence in the samples was confirmed by electrical effective capacitance measurements executed on the biosensor, and correlated with real-time PCR results. We verified that the biosensor can distinguish samples that are positive for the virus from those that are negative in a total of 7 positive and 16 negative samples.
In addition, the biosensor can be used for semi-quantitative measurement, since its measurements are divided into 3 areas, the negative samples, the weakly positive and the positive samples. Reproducibility of the experiments was demonstrated with at least 3 replicates and stability was tested by keeping the sensor standby for 7 days at 4 °C before repeating the experiment. This work presents a biosensor that can be used as a fast-screening test at point of care detection of SARS-CoV-2 since it needs less than 2 min to provide results and is of simple operation.

High-resolution melting PCR analysis for genotyping the gene polymorphism of TNF-α, TGF-β1, IL-10, and IFN-γ in lung transplant recipients

Background: High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation.

Objectives: This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs).

Material and methods: High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-β1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit.

Results: The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-β1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs.

Conclusions: In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.

A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex.

To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR.

Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

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Lanthanide luminescent nanocomposite for non-invasive temperature monitoring in vivo

February 13, 2022 Lieven Leave a comment

The temperature monitoring in vivo plays a vital role in the investigation of biological processes of organisms and the improvement of disease theranostic methods. The development of lanthanide luminescent nanocomposite-derived temperature probes in vivo allows accurate and reliable interrogation of biological thermodynamic processes due to their superior photostability, high sensitivity, and non-invasive sensing fashion.

This concept presented an overview of the recent development of lanthanide luminescent nanocomposite which are suitable for in vivo temperature monitoring, including the thermometric principles, key features, materials designs as well as their potential biomedical applications for non-invasive temperature detection in the living body. The perspectives of these lanthanide luminescent nanocomposite thermometers https://biodas.org/ for the optimization of temperature monitoring performance and potential future development are also discussed.

Habitat Use and Activity Patterns of Mammals and Birds in Relation to Temperature and Vegetation Cover in the Alpine Ecosystem of Southwestern China with Camera-Trapping Monitoring

The high-altitude ecosystem of the Tibetan Plateau in China is a biodiversity hotspot that provides unique habitats for endemic and relict species along an altitudinal gradient at the eastern edge. Acquiring biodiversity information in this area, where the average altitude is over 4000 m, has been difficult but has been aided by recent developments in non-invasive technology, including infrared-triggered camera trapping.

We used camera trapping to acquire a substantial number of photographic wildlife records in Wolong National Nature Reserve, Sichuan, China, from 2013 to 2016. We collected information of the habitat surrounding the observation sites, resulting in a dataset covering 37 species and 12 environmental factors. We performed a multivariate statistical analysis to discern the dominant environmental factors and cluster the mammals and birds of the ecosystem in order to examine environmental factors contributing to the species’ relative abundance. Species were generalized into three main types, i.e., cold-resistant, phyllophilic, and thermophilic, according to the identified key environmental drivers (i.e., temperature and vegetation) for their abundances.

The mammal species with the highest relative abundance were bharal (Pseudois nayaur), Moupin pika (Ochotona thibetana), and Himalayan marmot (Marmota himalayana). The bird species with highest relative abundance were snow partridge (Lerwa lerwa), plain mountain finch (Leucosticte nemoricola), Chinese monal (Lophophorus lhuysii), and alpine accentor (Prunella collaris).

Intraluminal Esophageal Temperature Monitoring Using the Circa S-Cath™ Temperature Probe to Guide Left Atrial Ablation in Patients with Atrial Fibrillation

Introduction: Radiofrequency catheter ablation is a common treatment for atrial fibrillation (AF), during which thermal esophageal injury may rarely occur and lead to an atrio-esophageal fistula. Therefore, we studied the utility of the Circa S-Cath™ multi-sensor luminal esophageal temperature (LET) probe to prevent esophageal thermal injury.

Methods and results: Thirty-six patients, enrolled prospectively, underwent circumferential or segmental pulmonary vein isolation for treatment of AF. A maximum ablation electrode temperature of 42ºC was programmed for automatic power delivery cutoff. In addition, energy delivery was manually discontinued when the maximum LET on any sensor of the probe rose abruptly (i.e. ˃0.2ºC) or exceeded 39º C. Esophagoscopy was performed immediately after ablation in 18 patients (with the temperature probe still in place) and at approximately 24 hours after ablation in 18 patients.

Esophageal lesions were classified as likely traumatic or thermally related. Of the 36 patients enrolled in the study, 21 had persistent and 15 had paroxysmal AF, average LVEF 57±16% and CHA2DS2VASc score 1.6±1.2 (range 0-4). Average maximum LET was 37.8±1.4ºC, power delivery 31.1±8 watts and ablation electrode temperature 36.4±4.1ºC. Average maximum contact force was 44.5±20.5 grams where measured. Only 1 patient (<3%) had an esophageal lesion that could potentially represent thermal injury and 4 patients (11.1%) had minor traumatic mechanical injury.

Conclusions: LET guided titration of power and duration of energy application, using an insulated multi-sensor esophageal temperature probe, is associated with a low risk of esophageal thermal injury during AF ablation. In only rare cases, LET monitoring resulted in the need to manipulate the esophagus to avoid unacceptable temperature rises, that could not be achieved by adjustment of power and duration of energy application.

Bio-Inspired Microwave Modulator for High-Temperature Electromagnetic Protection, Infrared Stealth and Operating Temperature Monitoring

High-temperature electromagnetic (EM) protection materials integrated of multiple EM protection mechanisms and functions are regarded as desirable candidates for solving EM interference over a wide temperature range. In this work, a novel microwave modulator is fabricated by introducing carbonyl iron particles (CIP)/resin into channels of carbonized wood (C-wood). Innovatively, the spaced arrangement of two microwave absorbents not only achieves a synergistic enhancement of magnetic and dielectric losses, but also breaks the translational invariance of EM characteristics in the horizontal direction to obtain multiple phase discontinuities in the frequency range of 8.2-18.0 GHz achieving modulation of reflected wave radiation direction.

Accordingly, CIP/C-wood microwave modulator demonstrates the maximum effective bandwidth of 5.2 GHz and the maximum EM protection efficiency over 97% with a thickness of only 1.5 mm in the temperature range 298-673 K. Besides, CIP/C-wood microwave modulator shows stable and low thermal conductivities, as well as monotonic electrical conductivity-temperature characteristics, therefore it can also achieve thermal infrared stealth and working temperature monitoring in wide temperature ranges. This work provides an inspiration for the design of high-temperature EM protection materials with multiple EM protection mechanisms and functions.

Non-Invasive Microwave Hyperthermia and Simultaneous Temperature Monitoring with a Single Theranostic Applicator

Cancer therapies are constantly evolving. Currently, heating tumor tissue is becoming more accessible as a stand-alone method or in combination with other therapies. Due to its multiple advantages over other heating mechanisms, microwave hyperthermia has recently gained a lot of traction.
In this work, we present a complementary split-ring resonator that is simultaneously excited in two independent frequency bands. With a high-power signal, the applicator is excited and heats the tissue-under-test up to 50°C with an average heating rate of 0.72°C per second. Furthermore, we present a dielectric temperature control system using the same applicator for microwave hyperthermia applications, which currently still requires an additional thermometry system. By exciting the applicator with a low-power signal, we can constantly monitor its resonant frequency.
This resonant frequency depends on the tissue properties, which in turn are temperature-dependent. In the temperature range from 20-50°C, a positive correlation between the temperature and resonant frequency was established.Clinical relevance – Exploiting the dual-band behavior of the complementary split-ring resonator to heat the tissue-under-test while dielectrically monitoring its temperature, creates new possibilities towards a theranostic, non-invasive microwave hyperthermia applicator.

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

February 13, 2022 Lieven Leave a comment

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 pK_a 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 https://biodas.org/ 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.

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A microfluidic DNA library preparation platform for next-generation sequencing

May 6, 2020 Lieven Leave a comment

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

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 (NGS) library 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%.