PicoGreen assay for nucleic acid quantification - Applications, challenges, and solutions.

Various analytical methods and reagents have been employed for nucleic acid analysis in cells, biological fluids, and formulations. Standard techniques like gel electrophoresis and qRT-PCR are widely used for qualitative and quantitative nucleic acid analysis. However, these methods can be time-consuming and labor-intensive, with limitations such as inapplicability to small RNA at low concentrations and high costs associated with qRT-PCR reagents and instruments. As an alternative, PicoGreen (PG) has emerged as a valuable method for the quantitative analysis of nucleic acids. PG, a fluorescent dye, enables the quantitation of double-stranded DNA (dsDNA) or double-stranded RNA, including miRNA mimic and siRNA, in solution. It is also applicable to DNA and RNA analysis within cells using techniques like FACS and fluorescence microscopy. Despite its advantages, PG's fluorescence intensity is affected by various experimental conditions, such as pH, salts, and chemical reagents. This review explores the recent applications of PG as a rapid, cost-effective, robust, and accurate assay tool for nucleic acid quantification. We also address the limitations of PG and discuss approaches to overcome these challenges, recognizing the expanding range of its applications.

Synthesis and Characterizations of MoS2 Nanoflowers and Spectroscopic Study of their Interaction with Bovine Serum Albumin.

Molybdenum disulfide nanoflowers (MoS2 NFs) were prepared by hydrothermal method. The prepared MoS2 NFs was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), specific surface areas, Raman and X-ray photoelectron spectroscopy (XPS). The characterization results show that the flower-like spherical MoS2 is composed of many ultra-thin nanosheets with an average diameter of about 300-400 nm. MoS2 NFs also exhibits excellent UV-vis absorption and high fluorescence intensity. In order to explore the biological behavior of MoS2 NFs, the interaction between MoS2 NFs and bovine serum albumin (BSA) was studied by UV-Vis absorption, fluorescence, synchronous fluorescence spectra, and cyclic voltammetry. The results of absorption and fluorescence show that MoS2 NFs and BSA interact strongly through the formation of complexes in the ground state, and the static quenching is the main mechanism. The Stern-Volmer constant and the quenching constant was calculated about 3.79×107 L mol-1 and 3.79×1015 L mol-1 s-1, respectively. The synchronous fluorescence implied that MoS2 in the complex may mainly bind to tryptophan residues of BSA. The cyclic voltammograms indicated that the addition of BSA makes electron reduction of MoS2 NFs more difficult than the corresponding free state. The results show that hydrophobic forces play a major role in the binding interaction between BSA and MoS2 NFs.

Preparation and Performance of a Fiber Optic Temperature Sensor with Multiple Fluorescence Mechanisms.

The tip of a piece of plastic fiber was dyed with thymol blue to form a temperature probe. The fiber optic sensor was calibrated on a heatboard by comparison with a K-type thermal couple. Fluorescence characteristics including fluorescence intensity, emission bandwidth, peak & barycenter wavelengths, and self-referenced intensity ratio were used to carry the information of environment temperature. Accordingly, more than five temperature sensing functions were retrieved from the fluorescent sensor. Among such functions, the emission band barycenter showed premium precision. Temperature-driven shift of the emission band barycenter has a sensitivity of 0.095 nm/K, with a nonlinearity of 2.2%FS, resolution of 4 K and repeatability of 1.8%FS. The sensor can find its applications in wearable devices and radiofrequency ablation. Finally in a verification experiment, the sensor was used to monitor the temperature of a microwave oven chamber in real time.

A novel optimized eco-friendly simple spectrofluorimetric method for the determination of total catechins in green tea extract: Application to commercial tablet.

Green tea extract (GTE) contains antioxidants that are present in green tea. The active constituents of green tea extract are catechins. This study demonstrates a spectrofluorimetric method for measuring GTE's catechin concentration based on its native fluorescence. To design a quick, sensitive, and ecological spectrofluorimetric approach, all features were investigated and adjusted. This method relies on determining the GTE ethanolic solution's native fluorescence at 312 nm after excitation at 227 nm. The calibration graph displayed a linear regression for values between 0.05 and 1.0 µg mL-1. The detection and quantification limits of the proposed technique were 0.008 and 0.026 µg mL-1, respectively. Two pure catechins present in GTE, (-)-epicatechin and (-)-epigallocatechin gallate, were examined by the proposed method. The analytical estimation of GTE in the pharmaceutical tablet was achieved effectively using this approach. An adequate degree of agreement was found when the findings were compared to those obtained by the comparative technique. Therefore, the novel strategy may be used in the GTE quality control study with minimal risks to people or the environment. The quantum yields of catechins were estimated. The validated technique was accepted by the International Council of Harmonization criteria.

Immunofluorescence microscopy of G-quadruplexes and R-loops.

A large variety of non-B secondary structures can be formed between DNA and RNA. In this chapter, we focus on G-quadruplexes (G4) and R-loops, which can have a close structural interplay. In recent years, increasing evidence pointed to the fact that they can strongly influence each other in vivo, both having physiological and pathological roles in normal and cancer cells. Here, we detail specific and accurate methods for purification of BG4 and S9.6 antibodies, and their subsequent use in immunofluorescence microscopy, enabling single-cell analysis of extent and localization of G4s and R-loops.

Time-resolved fluorescence and diffuse reflectance for lung squamous carcinoma margin detection.

OBJECTIVES: A major challenge in non-small cell lung cancer surgery is the occurrence of positive tumor margins. This may lead to the need for additional surgeries and has been linked to poor patient prognosis. This study aims to develop an in vivo surgical tool that can differentiate cancerous from noncancerous lung tissue at the margin. METHODS: A time-resolved fluorescence and diffuse reflectance bimodal device was used to measure the lifetime, spectra, and intensities of endogenous fluorophores as well as optical properties of lung tissue. The tumor and fibrotic tissue data, each containing 36 samples, was obtained from patients who underwent surgical removal of lung tissue after being diagnosed with squamous carcinoma but before any other treatment was administered. The normal lung tissue data were obtained from nine normal tissue samples. RESULTS: The results show a statistically significant difference between cancerous and noncancerous tissue. The results also show a difference in metabolic related optical properties between fibrotic and normal lung tissue samples. CONCLUSIONS: This work demonstrates the feasibility of a device that can differentiate cancerous and noncancerous lung tissue for patients diagnosed with squamous cell carcinoma.

A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson's Disease.

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson's disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

Novel phosphor GdY2SbO7 co-dope with Eu3+ and Bi3+ for optical thermometer.

A series of GdY2SbO7:Bi3+, Eu3+ phosphors were prepared using the conventional solid-state reaction. In this study, the photoluminescence properties and temperature sensitivity of the samples were investigated. When Bi3+ and Eu3+ were codoped into GdY2SbO7, the intensity of Bi3+ decreased with increasing Eu3+ concentration, indicating a potential energy transfer from Bi3+ to Eu3+. To examine the temperature sensitivity of the sample, its emission spectrum was investigated in the range of 300-500 K. Based on different temperature dependences of Bi3+ and Eu3+, the relative sensitivity (Sr) and absolute sensitivity (Sa) of the samples were calculated using the fluorescence intensity ratio (FIR) and thermochromic methods. In FIR modes, Sr based on IEu3+/IBi3+ reached 1.26 % K-1 at 500 K, while Sr on double excitation method reached 1.36 % K-1 at 340 K. In addition, according to the thermochromic properties of GdY2SbO7:Bi3+, Eu3+ phosphor, the temperature-sensing ability of the sample was investigated, and Sr reached a maximum value of 0.5996 % K-1 at 300 K.

Use of Super-Resolution Live Cell Imaging to Distinguish Endoplasmic Reticulum: Nuclear Envelope Subcellular Localization.

A distinguishing feature of eukaryotes is the presence of a nuclear envelope (NE) and endomembrane system. The NE is a double-membrane system that surrounds chromatin and is continuous with the endoplasmic reticulum (ER). This interface is crucial in various processes such as calcium signaling and ER-associated degradation. The outer nuclear membrane and ER share a multitude of proteins although some are only functional in one domain, whereas the inner nuclear membrane has its own unique proteome. Until recently, it was not possible to distinguish between the inner and outer nuclear membranes as well as perinuclear ER using light microscopy - only electron microscopy was suitable for this. Now, however, using super-resolution live cell imaging, this can be achieved while still observing protein and membrane dynamics in real time. The protocols described here will allow researchers to determine subcellular localization of potential NE/ER proteins in live plant cells, helping to gain new insights into protein functionality.

Li4 Zn(PO4 )2 :Mn2+ thermosensitive phosphor with dual luminescent centres for optical thermometry.

An optical thermometry strategy based on Mn2+ -doped dual-wavelength emission phosphor has been reported. Samples with different doping content were synthesized through a high-temperature solid-phase method under an air atmosphere. The electronic structure of Li4 Zn(PO4 )2 was calculated using density functional theory, revealing it to be a direct band gap material with an energy gap of 4.708 eV. Moreover, the emitting bands of Mn2+ at 530 and 640 nm can be simultaneously observed when using 417 nm as the exciting wavelength. This is due to the occupation of Mn2+ at the Zn2+ site and the interstitial site. Further analysis was conducted on the temperature-dependent emission characteristics of the sample in the range 293-483 K. Mn2+ has different responses to temperature at different doping sites in Li4 Zn(PO4 )2 . Based on the calculations using the fluorescence intensity ratio technique, the maximum relative sensitivity at a temperature of 483 K was determined to be 1.69% K-1 , while the absolute sensitivity was found to be 0.12% K-1 . The results showed that the Li4 Zn(PO4 )2 :Mn2+ phosphor has potential application in optical thermometry.

Temperature Effect on Fluorescence Intensity and Dipole Moment Using Thermochromic Shift Method of 7DA3MHBI-2HChromen-2-one Laser Dye in Highly Viscous Glycerol Solvent.

The steady-state method is used to study the effect of temperature on the fluorescence characteristics of 7-(diethylamino)-3-(1-methyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7DA3MHBI-2HChromen-2-one) laser dye in glycerol solvent for the temperature range 293-343 K. Absorption and emission characteristics are affected by varying temperatures due to induced thermal effects. Transition probabilities mechanism of non-radiative and radiative are studied and frequency dependent parameters are estimated. Dipole moments in the ground and excited state are estimated using the thermochromic shift method over general solvatochromic methods.

Encoding fluorescence intensity with tetrahedron DNA nanostructure based FRET effect for bio-detection.

Biocoding technology constructed by readable tags with distinct signatures is a brand-new bioanalysis method to realize multiplexed identification and bio-information decoding. In this study, a novel fluorescence intensity coding technology termed Tetra-FICT was reported based on tetrahedron DNA nanostructure (TDN) carrier and Forster Resonance Energy Transfer (FRET) effect. By modulating numbers and distances of Cy3 and Cy5 at four vertexes of TDN, different fluorescence intensities of twenty-six samples were produced at ∼565.0 nm (FICy3) and ∼665.0 nm (FICy5) by detecting fluorescence spectra. By developing an error correction mechanism, eleven codes were established based on divided intensity ranges of the final FICy3 together with FICy5 (Final-FICy3&FICy5). These resulting codes were used to construct barcode probes, with three miRNA biomarkers (miRNA-210, miRNA-199a and miRNA-21) as cases for multiplexed bio-assay. The high specificity and sensitivity were also demonstrated for the detection of miRNA-210. Overall, the proposed Tetra-FICT enriched the toolbox of fluorescence coding, which could be applied to multiplexing biomarkers detection.

Label-free fluorescence aptasensor for the detection of cadmium(II) ion based on the conformational switching of aptamer and thioflavine T.

A simple label-free Cd2+ fluorescent aptasensor was proposed using aptamer as a recognition element and thioflavine T (ThT) as a signal reporter. The presence of Cd(II) can induce the conformational switching of the aptamer probe, accompanied by a change in fluorescence intensity. According to the difference in fluorescence signals before and after the addition of Cd2+, a fluorescence sensor for Cd2+ assay was established. Under the better experimental conditions, the sensor displayed a good linear range from 2 to 50 nM and the excellent detection limit was 0.8 nM. The method demonstrated high sensitivity and good selectivity. The aptasensor could detect Cd2+ in simulated water samples with satisfactory results. The proposed method had obvious advantages that was without chemical modification of fluorescent groups and complicated target preconcentration. It provided a new analytical platform for the detection of heavy metal ion pollution in environmental and biomedical fields.

Fluorescent Properties of Cyanine Dyes As a Matter of the Environment.

In non-viscous aqueous solutions, the cyanine fluorescent dyes Cy3 and Cy5 have rather low fluorescence efficiency (the fluorescence quantum yields of Cy3 and Cy5 are 0.04 and 0.3, respectively [1, 2]) and short excited state lifetimes due to their structural features. In this work, we investigated the effect of solubility and rotational degrees of freedom on the fluorescence efficiency of Cy3 and Cy5 in several ways. We compared the fluorescence efficiencies of two cyanine dyes sCy3 and sCy5 with the introduction of a sulfonyl substituent in the aromatic ring as well as covalently bound to T10 oligonucleotides. The results show that because of the different lengths of the polymethine chains between the aromatic rings of the dyes, cis-trans-isomerization has a much greater effect on the Cy3 molecule than on the Cy5 molecule, while the effect of aggregation is also significant.

Harmonization of ANA testing challenge: quantification strategy to accurately predict end-point titers avoiding serial dilution.

Despite the advantages of automated systems for antinuclear antibody (ANA) analysis, the prediction of end-point titers avoiding serial dilutions is still in progress. The aims of this study were to set a conversion table providing discriminant ranges of fluorescence signal intensity values (FI) corresponding to the end-point titers and validate this tool in a real-life laboratory setting. Eight hundred ninety-four serum samples were analyzed for ANA using Image Navigator System. In order to classify FI into non-overlapping groups corresponding to conventional end-point titers, statistical discriminant analysis was used. Validation study was performed calculating agreement and error rates between visual readings and conversion table of 1119 routine ANA positive samples. Setting of FI ranges corresponding to the end-point titers for different staining patterns was computed. For samples showing single pattern, the overall agreement between visual readings and conversion table was 98.4% for all titers ranging from 1:160 to 1:2560, of which 68.0% had the same titer and 30.4% were within ± one titer difference. Concordance rates according to ANA patterns were as follows: (1) nuclear 98.4%, of which 67.0% had the same titer and 31.4% ± one titer; (2) cytoplasmic 100%, of which 72.7% had the same titer and 27.3% than ± one titer; (3) mitotic 66.6%, of which 33.3% had more ± one titer. Our study developed a quantification method for autoantibodies titers assessment based on just one single sample dilution instead of traditional serial dilution approach, providing significant advantages in routine laboratory in terms of reduction in hand-on time and harmonization of results.

Maillard Reaction Process and Characteristic Volatile Compounds Formed During Secondary Thermal Degradation Monitored via the Change of Fluorescent Compounds in the Reaction of Xylose-Corn Protein Hydrolysate.

Until now, no effective method has been found to monitor the Maillard reaction process for complex protein hydrolysates. Dynamic changes in the concentration of α-dicarbonyl compounds, fluorescence intensity, and browning degree were investigated during the Maillard reaction of corn protein hydrolysates. When the fluorescence intensity reached the peak, deoxyosones would continue to be increased by ARP's degradation. However, the reaction node with the highest fluorescence intensity coincided with the turning point of the browning reaction, and the subsequent browning rate remarkably increased. Therefore, the change in fluorescence intensity could be used to monitor the degradation of ARP and the formation of browning melanoidin at different stages of the Maillard reaction of complex systems, thus effectively indicating the process of the Maillard reaction. When Maillard reaction intermediates (MRIs) with maximum fluorescent compounds were heated, the most abundant pyrazines were subsequently achieved. However, furan compounds would be progressively increased during the thermal process of MRIs with continuously enhanced browning.

Surface Functionalised Optical Fibre for Detection of Hydrogen Sulphide.

Dysregulated production of hydrogen sulphide in the human body has been associated with various diseases including cancer, underlining the importance of accurate detection of this molecule. Here, we report the detection of hydrogen sulphide using fluorescence-emission enhancement of two 1,8-naphthalimide fluorescent probes with an azide moiety in position 4. One probe, serving as a control, featured a methoxyethyl moiety through the imide to evaluate its effectiveness for hydrogen sulphide detection, while the other probe was modified with (3-aminopropyl)triethoxysilane (APTES) to enable direct covalent attachment to an optical fibre tip. We coated the optical fibre tip relatively homogeneously with the APTES-azide fluorophore, as confirmed via x-ray photoelectron spectroscopy (XPS). The absorption and fluorescence responses of the control fluorophore free in PBS were analysed using UV-Vis and fluorescence spectrophotometry, while the fluorescence emission of the APTES-azide fluorophore-coated optical fibres was examined using a simple, low-cost optical fibre-based setup. Both fluorescent probes exhibited a significant increase (more than double the initial value) in fluorescence emission upon the addition of HS- when excited with 405 nm. However, the fluorescence enhancement of the coated optical fibres demonstrated a much faster response time of 2 min (time for the fluorescence intensity to reach 90% of its maximum value) compared to the control fluorophore in solution (30 min). Additionally, the temporal evolution of fluorescence intensity of the fluorophore coated on the optical fibre was studied at two pH values (7.4 and 6.4), demonstrating a reasonable overlap and confirming the compound pH insensitivity within this range. The promising results from this study indicate the potential for developing an optical fibre-based sensing system for HS- detection using the synthesised fluorophore, which could have significant applications in health monitoring and disease detection.

Fallacies of a purely virtual platform: Virtual plus reality versus virtual reality - A case study.

Pretransplant immunological assessment of a transplant donor has evolved significantly over the last few decades with the advent of testing platforms with enhanced sensitivity and varying formats. The single antigen bead assay (SAB) assay, a virtual crossmatch (vXM) is used extensively and considered the gold standard for defining donor-specific antibodies (DSA) in many parts of the World. A country like India, is however challenged by the lack of adequate representation of locally frequent HLA alleles and hence in our institution, we continue to perform a physical crossmatch (pXM) on the Complement Dependent Cytotoxicity and flow cytometry platforms alongside the SAB. We report here a case report where the discrepancy between platforms of testing have raised certain pertinent questions in our interpretation of the vXM.

Anti-Hla Donor-Specific Antibodies Are Associated to Infection and Not to the Engraftment Rate in Outpatient Haploidentical Hematopoietic Cell Transplantation.

Background: Recipients of a related haploidentical stem cell transplant (haplo-SCT) can have preformed antibodies to HLA donor's antigens. Objective: The aim of the study was to evaluate the engraftment rate and major clinical associations of anti-HLA donor-specific antibodies (DSA) at two mean fluorescence intensity (MFI) thresholds in recipients of an outpatient haplo-SCT. Methods: Seventy haplo-HCT recipients were analyzed. A virtual crossmatch was performed using the donor HLA typing and the recipient's anti-HLA DSA test results. Data for anti-HLA-A, -B, -C, and -DR were analyzed. Recipients with DSA ≥ 500 MFI were considered positive, and those with < 500 were considered negative; the same was adopted for MFI ≥ 1000. Results: Post-transplant infection was higher in recipients with DSA ≥ 500 MFI (84.6%, p = 0.041). First-year mortality was higher in DSA-positive patients ≥ 500 MFI, p = 0.004, and DSA ≥ 1000 MFI, p = 0.022, than in DSA-negative recipients. Graft failure in the first 100 days was not associated with DSA ≥ 500 or ≥ 1000 MFI. There was no difference in acute (a-GVHD) or chronic (c-GVHD) graft versus host disease between DSA-positive and negative patients. Conclusions: There was no association of anti-HLA DSA at MFI ≥ 500 and ≥ 1000 with graft failure, however, increased infection and 1st-year mortality were documented in related haplo-HCT at the MFI cutoffs studied.

Synthesis and performance analysis of zeolitic imidazolate frameworks for CO2 sensing applications.

In this paper, we investigate the potential use of Zeolitic Imidazolate Frameworks (ZIF-8) as a sensing material for CO2 detection. Three synthesis techniques are considered for the preparation of ZIF-8, namely room temperature, microwave-assisted, and ball milling. The latter is a green and facile alternative for synthesis with its solvent-free, room-temperature operation. In addition, ball milling produces ZIF-8 samples with superior CO2 adsorption and detection characteristics, as concluded from fluorescence measurements. Characterization tests including X-ray diffraction (XRD), Fourier transform infrared (FTIR), Thermogravimetric analysis (TGA), Field emission scanning electron microscopy (FE-SEM) and Energy-dispersive X-ray spectroscopy (EDS) are conducted to inspect the structural morphology, the thermal stability, and elements content of the ZIF-8 samples obtained from the different aforementioned synthesis techniques. The characterization tests revealed the appearance of a new phase of ZIF-8 which is ZIF-L when deploying the ball milling technique with different structure, morphology, response to CO2 exposure and thermal stability when compared to its counterparts. Fluorescence measurements are carried out to evaluate the limit of detection (LOD), selectivity, and recyclability of the different ZIF-8 samples. The LOD of the ZIF-8 sample synthesized based on ball milling synthesis technique is 815.2 ppm, while LODs of the samples obtained from microwave and room temperature-based synthesis techniques are 1780.6 ppm and 723.8 ppm, respectively. This indicates that the room temperature and ball milling produced MOFs have comparable LODs. However, the room temperature procedure requires the use of a harmful solvent. The range of LOD demonstrates the suitable use of ZIF-8 for indoor air quality monitoring and other industrial applications.