HEPES Buffer Research Articles

Highly sensitive label-free liquid crystal-based aptasensor to detect alpha-fetoprotein

ABSTRACT In this study, a novel label-free optical aptasensor based on liquid crystals (LCs) for sensitive and specific alpha-fetoprotein (AFP) detection was developed. The modification parameters were optimised to limit the effect of the background signal and increase the sensitivity of the sensor. The AFP-specific aptamer was immobilised on the substrates using an APTES + GA bridge. The binding of AFP to its aptamer causes changes in the surface topography and disrupts the LC alignment. The orientational transition of LC could be observed, from dark to bright, using POM. The changes in the topography of the surfaces were confirmed by AFM, ellipsometry, and water contact angle measurements. Under optimal conditions, the proposed LC-based aptasensor exhibited optimal performance in AFP detection. The detection limit for AFP reached 12.62 pg mL−1 in HEPES buffer and 18.65 pg mL−1 in human serum, with the detection range from 20–800 mL−1 for both the conditions. Owing to advantages such as simplicity, speed, and high sensitivity and specificity, the proposed label-free LC-based aptasensor has potential applications in the clinical diagnosis of various tumour markers.

A 3D Bioprinted Human Meniscus Shape Enriched with Mesenchymal Cells

Background and objectives: Regenerative medicine, with its massive development over the years, has the potential to solve some of the most problematic medical issues, such as functional organ transplantation. The aim of this study was to create a human meniscal shape 3D-printed enriched with human adipose-derived mesenchymal cells. Materials and Methods: Human infrapatellar fat pad was harvested, and mesenchymal cells were isolated. The mesenchymal stem cells were differentiated to the chondrocite lineage and a hydrogel (a nanofibrillar cellulose, sodium alginate, D-mannitol, and Hepes buffer solution combination) cell mixture was bioprinted to create three human-size meniscus structures. The obtained structures were evaluated regarding the cell viability, appropriate size in relation to a native meniscus, and some mechanical characteristics. Results: The human meniscal shape created respected the anatomic characteristic of a native structure. Cell viability of approximately 97% and extracellular matrix formation after the printing process were observed. The mean maximum force for the meniscus with mesenchymal cells was 6.5 N (+/−0.5 N) compared to the mean maximum force for the native meniscus of 10.32 N (+/−0.7 N), which is statistically relevant (p < 0.01). Conclusion: This paper presents the potential of bioprinting viable cell structures that could in the future present enough mechanical strength to replace a human organ, such as a meniscus. There are still limitations regarding the ink and the printing process, but we are confident that these problems will soon be solvable.

Assessment of biological activity of the caffeine‐derived Pt (II) and Pd (II) complexes

AbstractThe substitution reactions of [Pd (caffeine)2Cl2] and [Pt (caffeine)2Cl2] (caffeine = 1,3,7‐trimethylxanthine) complexes with bio‐molecules such as 9‐methylguanine (9‐MetGua) and guanosine‐5′‐monophosphate (5′‐GMP) were studied spectrophotometrically. Kinetic measurements were performed under the pseudo‐first‐order conditions at 37°C and pH = 7.2 (25‐mM Hepes buffer) with the addition of 50‐mM NaCl. All reactions were carried out in two reaction steps giving the [M (caffeine)2(Nu)2] (M = Pd (II) or Pt (II) and Nu = 5′‐GMP or 9‐MetGua) as the reaction product. The reaction mechanism was also confirmed by nuclear magnetic resonance (NMR) spectroscopy and density functional theory (DFT) calculations. Kinetically data revealed a much higher reactivity of [Pd (caffeine)2Cl2] complex compared with analog platinum‐complex, while 9‐MetGua reacted faster than 5′‐GMP. The interactions of [Pd (caffeine)2Cl2] and [Pt (caffeine)2Cl2] complexes with calf thymus‐DNA (CT‐DNA) and human serum albumin (HSA) were investigated as well. Competitive studies with DNA were performed in the presence of ethidium bromide (EB) and Hoechst dye 33258 (Hoe). The complexes interact with DNA via minor groove rather than by intercalation. High values of binding constants indicate a good binding affinity of complexes toward HSA (104 M−1). Additionally, the experimental results of binding studies between [Pt (caffeine)2Cl2] complex with DNA and HSA were compared by a molecular docking study. The cytotoxicity of [Pd (caffeine)2Cl2] and [Pt (caffeine)2Cl2] complexes was tested against the mouse breast cancer (4T1) and colon cancer (CT26) as well as the human breast cancer (MDA‐MB‐468) and colon cancer (HCT‐116)] cell lines. The results indicate that the [Pt (caffeine)2Cl2] complex has higher cytotoxic capacity compared with [Pd (caffeine)2Cl2] at concentrations higher than 2 μM.

Double advantages of Benzimidazolyl‑hydroxy-coumarinyl diad - “turn-on” sensing of Al3+ and preventing metastasis in cancer

8-(1H-Benzoimidazol-2-yl)-7‑hydroxy-4-methyl-chromen-2-one (HL), a benzimidazolyl‑hydroxy-coumarinyl diad, serves as fluorescent turn-on Al3+-sensor in acetonitrile-water (2:1, v/v) of pH, 7.2 (HEPES buffer) medium. The intensity of emission on binding with Al3+ is enhanced by twelve times of probe with limit of detection (LOD) 0.62 µM. The structure of the probe has been established by the spectroscopic data (FT-IR, 1H NMR, Mass spectra) and is confirmed by Single Crystal X-ray structure determination. The co-ordination environment of the complex, [Al(L)(NO3)2(H2O)(NCCH3)], has been supported by Mass, FT-IR, NMR spectra and the Job's plot supports 1:1 composition. The anti-metastatic potential of HL on Human skin carcinoma (A431) and Human lung carcinoma (A549) cells are examined. Wound healing and Zymography assay suggest that HL has anti-migratory properties. The RT-PCR and Immunoblotting data suggest that HL down-regulates the expression of mesenchymal markers (Vimentin, Snail, Slug, MMP, and MMP9) and induces the expression of the epithelial marker (E-Cadherin).

The effect of nitric oxide synthase and arginine on the color of cooked meat

In previous studies, it has been suggested that the NO-synthase enzyme may be responsible for color formation in fermented sausages. Thus, this is the first study in which the aim was to analyze the effects of direct NO-synthase and arginine application to meat on its color after heating. Myoglobin forms as well as the presence of NO-myoglobin were investigated. The color of the meat and myoglobin forms present in the samples were mainly affected by pH differences, caused by a HEPES buffer or arginine. None of the variants demonstrated a bright pink color as in the case of the heated nitrite-cured sample. Based on analysis of the absorption spectra, it can be concluded that there is some evidence of nitroso-complex formation. Therefore, it is probable that optimizing the pH/time/temperature conditions for NO-synthase activity would allow to obtain a desirable color effect. NO-synthase could be used as an alternative curing ingredient.

Facile synthesis and electrochemical performance of lithium-rich layered oxides with stable hierarchical structure through HEPES-assisted co-precipitation method

Lithium-rich layered oxide (LLO) electrode material is now actively developed as a promising cathode for next-generation lithium-ion batteries due to its high specific capacity. The electrochemical property of the LLO material is mainly regulated by preparation conditions. Here, we report a facile 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) assisted co-precipitation method to prepare Li1.2Mn0.54Co0.13Ni0.13O2. The HEPES buffer solution with strong pH buffering capacity not only can keep the pH of the reaction solution stable, but also acts a complexing agent to promote the uniform distribution of elements and in control of the nucleation and growth rate of crystals during the co-precipitation process. The dispersant role of HEPES helps to prepare regular LLO microspheres with unique hierarchical structure, which endows sufficient contact of the particles with electrolyte, shortens Li+ diffusion path and provides high structural stability. The electrode demonstrates superior electrochemical performance and excellent cycling stability at different discharge currents. High discharge capacities of 147.2 and 100.0 mAh g−1 could be achieved after 200 cycles at high current densities of 5 C and 10 C, respectively. The paper provides a novel strategy of the controllable route for high energy storage materials and reveals the correlation between the cation distribution and the electrochemical performance stability of the materials.

Ultra-Fast Oxygen Monitoring During Proton FLASH Delivery Using the Phosphorescence Quenching Method and Soluble Oxyphor Probes

<h3>Purpose/Objective(s)</h3> FLASH is a promising form of radiation therapy due to its reported ability to spare normal tissues while being equipotent in tumor control. The mechanism underlying this tissue sparing effect is currently unknown, however the depletion of tissue oxygen at FLASH dose rates has been proposed as a potential source. To date, the oxygen depletion hypothesis has only been supported by analytical or numerical modeling studies, primarily due to the difficulty of measuring oxygen concentration on millisecond time scales during irradiation. In this project, we use the phosphorescence quenching method to probe oxygen depletion during proton delivery at both conventional (∼1Gy/sec) and FLASH (∼100 Gy/sec) dose rates. <h3>Materials/Methods</h3> Oxygen concentration was measured at rates of up to 3kHz using a phosphorescent probe, Oxyphor PtG4, and a time-domain OxyLED phosphorometer modified for ultra-fast operation. Sealed glass vials, containing the experimental solution and probe, were placed such that they were exposed to a uniform dose rate where the proton beam entered the vial. Radiation was delivered at conventional (conv) (∼1 Gy/s) and FLASH (∼100 Gy/s) dose rates. The g-value of oxygen depletion was measured as a function of dose rate, total dose, oxygen concentration, biomolecule concentration, and in the presence or absence of oxygen scavengers. Biomolecule relevant experiments were carried out using either 20mM phosphate buffer containing 1, 2, and 5% bovine serum albumin (BSA) or a solution designed to emulate the intracellular environment, containing 5mM glucose, 1M glycerol, 5mM glutathione, and 10 mM HEPES (Buffer 3G). Oxygen concentration was continuously monitored before, during, and after irradiation for all experiments. <h3>Results</h3> We found that FLASH dose rates resulted in lower g-values for oxygen depletion when compared to conventional dose rates by ∼15% (range: 7.2 to 22.5%) for each of the solutions tested. For example, in Buffer 3G, for a 30Gy dose, the g-value was 16% lower at FLASH dose rates (g_conv = 0.72 ± 0.02 μM/Gy, g_FLASH = 0.63 ± 0.02 μM/Gy) (all values listed are mean ± S.E.M.). The g-value increased with increasing BSA concentration (values for 1% and 5% BSA listed) at both conventional and FLASH dose rates (g_conv = 0.45 ± 0.05 and 0.58 ± 0.03 μM/Gy respectively, g_FLASH = 0.37 ± 0.01 and 0.62 ± 0.01 μM/Gy respectively). The g-value was higher for Buffer 3G than for the BSA solutions (g_conv = 0.72 ± 0.02 and 0.58 ± 0.03 μM/Gy respectively, g_FLASH = 0.63 ± 0.02 and 0.54 ± 0.01 μM/Gy respectively). An "after FLASH" period of gradual oxygen depletion which persisted for ∼0.5s and depleted an additional 32.1% oxygen was observed for Buffer 3G. <h3>Conclusion</h3> We have demonstrated a method for ultra-fast measurements of oxygen concentration during the delivery of proton radiation. Furthermore, we found that the g-value of oxygen depletion at FLASH dose rates is lower than that of conventional dose rates by approximately 15%.

A New Thiophene‐Appended Fluorescein‐Hydrazone‐Based Chromo‐Fluorogenic Sensor for the Screening of Hg2+ Ions in Real Water Samples

AbstractA simple yet efficient, Hg2+ selective sensor, based on a fluroescein‐thiophene conjugate, namely, (Z)‐3′,6′‐dihydroxy‐2‐(((3‐methylthiophen‐2‐yl)methylene)amino)spiro[isoindoline‐1,9′‐xanthen]‐3‐one (FT) has been designed and synthesized. The compound was well characterized by analytical techniques such as HR‐MS, FTIR, UV‐visible absorption, steady‐state fluorescence and time‐resolved spectrophotometric techniques. Comprehensive analysis demonstrated FTas a highly specific and ultra‐sensitive sensor towards Hg2+ ions in presence of several other interfering metal ions in ethanol/H2O (9 : 1, v/v) at pH=7.2 buffered with 10 mM HEPES buffer at room temperature. The complexation of the probe FT with Hg2+ was well confirmed by ESI‐MS and FTIR spectral analysis. The detection limit of the probe FT towards Hg2+ was achieved down to 137 nM. The structure of ligand FT and FT‐Hg2+ complex were well corroborated by theoretical studies as well. In addition, the FT‐Hg2+ complex was found to be reversible in presence of di‐sodium EDTA and hence decipher its recyclability capabilities. The significance of the present probe FT lies in its successful application for the detection and quantification of Hg2+ in real water samples and logic gate fabrication for future incorporation in small organic molecule based efficient molecular devices.

New mononuclear gold(III) complexes: Synthesis, characterization, kinetic, mechanistic, DNA/BSA/HSA binding, DFT and molecular docking studies

Five new gold(III) complexes, with general formula [Au(NN)Cl2]+ for complexes 1–3 and [Au(NN)2]3+ for complexes 4–5 (where NN is 3-((2-((5-phenyl-1H-pyrazol-3-yl)methoxy)naphthalene-3-yloxy)methyl)-5-phenyl-1H-pyrazole – L1, (3-((2-((5-phenyl-1H-pyrazol-3-yl)methoxy) phenoxy)methyl)-5-phenyl-1H-pyrazole – L2, (3-((2-((5-naphthalen-2-yl)-1H-pyrazol-3-yl)methoxy)phenoxy)methyl)-5-(naphthalen-3-yl)-1H-pyrazole – L3), were synthesized and characterized by elemental analysis, 1H NMR, IR, UV–Vis, ESI-MS and conductometry. Also, ligand L2 was characterized by X-ray analysis. Stability of complexes in water and in Hepes buffer was confirmed by UV–Vis. Kinetics and mechanism of the substitution reactions of 1–3 with guanosine-5′-monophosphate (5′-GMP), glutathione (GSH) and.l-Methionine (l-Met) were studied by stopped-flow technique. Obtained results have shown that complex 1 is the most reactive, while the reactivity of the nucleophiles decreases in order: GSH > 5′-GMP > l-Met. Calculated values of the entropy of activation support an associative mechanism. Redox stability of complexes 1–3 was investigated in the presence of the same biomolecules by cyclic voltametry. Obtained voltammograms showed reduction of gold(III) up to gold(0). DNA binding studies in the presence of ethidium bromide (EB) and 2-(4-hydroxyphenyl)-5-[5-(4-methylpipera-zine-1yl)-benzimidazo-2-yl]-benzimidazole (HOE) were performed by UV–Vis, fluorescence spectroscopy and viscosity measurements, in order to assess the binding mode. The results showed that gold(III) complexes interact with calf-thymus (CT-DNA) via covalently binding mode rather than via intercalation. Also, all complexes shown high values of binding constants for the interactions with bovine serum albumin (BSA) and human serum albumin (HSA). Furthermore, the binding studies with CT-DNA and BSA/HSA were supported by molecular docking. The strong connection between structure and reactivity of gold(III) complexes toward biologically important molecules was confirmed.

Irreversible coumarin based fluorescent probe for selective detection of Cu2+ in living cells

Copper ion (Cu2+) is an essential part of the living organisms. Cu2+ ions play a vital role in many biotic processes. An abnormal amount of Cu2+ ions may result in serious diseases. Herein, a novel “fluorescent ON” probe NC-Cu to trace minute levels of Cu2+ ions in presence of various biological active species has been developed. Lysosomal cells targeting group (Morpholine) was added to the probe. The spectral properties of probe NC-Cu were recorded in HEPES buffer (0.01 M, pH = 7.4, comprising 50% CH3CN, λex = 430 nm, slit: 5 nm). The synthesized probe NC-Cu work based on copper promoted catalytic hydrolysis of hydrazone and shows remarkable fluorescence enhancement. The reaction of the probe with Cu2+ ions was completed within 20 min. An excellent linear relationship (R2 = 0.9952) was found and the limit of detection (LOD, according to the 3σ/slope) for Cu2+ ions was calculated to be 5.8 µM. Furthermore, NC-Cu was effectively functional in the living cells (KYSE30 cells) to trace Cu2+ ions.

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death

Construction of enhanced fluorescence sensors in aqueous media by cation regulation and hybridization

For sensing application, a salicylacylhydrazone (H2L) with multiple binding sites which was condensed by salicylhydrazide and 4-acylpyrazolone, H2L = 4-Benzoyl-3-methyl-l-phenyl-5-pyrazolone-benzoic acid hydrazide was prepared. Fluorescence studies indicate that H2L could be used as turn-on sensors to distinguish between Cd2+ and Zn2+ in Hepes buffer solution (pH = 6.8) and the origin of the specificity of the fluorescence recognition was disclosed through 1H NMR spectra, UV–Vis spectra, ESI mass spectra, X-ray single crystal diffraction and DFT calculations. H2L test silica sheets were further prepared to offer easy and real-time detection of Cd2+ and Zn2+ ions. Furthermore, the in situ system generated from the sensing of Zn2+ and Cd2+ (ZnHL and CdHL) showed good relay recognition ability for picric acid (PA) via fluorescence quenching. The recognition mechanism of PA could be attributed to the multi-quenching mechanism dominated by the competitive absorption with PET effect and hydrogen bond being assistance when density functional theory calculations and multiple experimental evidences, including ESI mass spectrum, 1H NMR spectra as well as UV–Vis absorption are combined. To enhance their sensing ability together with the convenience of detection, the hybrid films of ZnHL@PMMA and CdHL@PMMA are prepared by doping them to polymethyl methacrylate (PMMA) matrix to improve their fluorescence performance, chemical stability, sensitivity, cheapness and quickness.

Colorimetric chemosensor for Cu(II) from electrospun nanofibrous mat mixed with 5‐methoxy‐salicylaldehyde azine

AbstractThe condensation product of 5‐methoxy‐salicylaldehyde with hydrazine hydrate (referred to below as compound 1) was used as a ligand in a cellulose acetate electrospun nanofibrous mat to determine copper ions in aqueous solutions. The electrospun nanofibrous mats, based on cellulose acetate mixed with compound 1, were constructed using an electrospinning method and had a diameter of 260 nm. The sensing nanofibrous mat exhibited a sensitive and selective response for Cu(II) ions over a linear concentration range of 1‐30 µM in a 0.1 M HEPES buffer (pH 7.4) using a colorimeter. The detection limit of the method was 1.04 µM lower than the maximum level of Cu(II) in drinking water (16 µM, as determined by the US Environmental Protection Agency). Additionally, it exhibited high selectivity for Cu(II) ions over 13 other cations. The sensing nanofibrous mat was fully reversible for ethylenediamine tetraacetic acid treatment cycles. Preliminary results indicated the sensitivity and selectivity of the nanofibrous mat for detecting Cu(II) ions by naked‐eye detection from yellow to orange. The reaction product of compound 1 and the Cu(II) ion was studied using X‐ray absorption spectroscopy. The Cu‐coordination number was approximately 5.023 ± 1.42, and the Cu–N distance (R) was 1.986 Å. The nanofibrous mat demonstrated a convenient and straightforward Cu(II) ion‐detection technique in both wastewater and water environments.

Using Zn and Ni behavior during magnetite precipitation in banded iron formations to determine its biological or abiotic origin

There is longstanding controversy about the genesis of magnetite in banded iron formations (BIFs), particularly concerning whether it is of abiogenic or biogenic origin. The composition of trace elements within magnetite produced by magnetotactic bacteria has been proposed as a promising marker for its biological origin; however further experimental evidence is required to investigate whether this also holds true for iron-metabolizing bacteria. Here, we compared the behavior of zinc (Zn) and nickel (Ni) in magnetite produced either abiogenically (ferrihydrite reacting with dissolved Fe2+) or biogenically (ferrihydrite reacting with dissolved Fe2+ generated via the dissimilatory iron-reducing bacterium - DIRB - Shewanella oneidensis MR-1). These abiotic and biotic incubations were applied for transforming three ferrihydrite (Fh) substrates: (1) Control Fh without added trace elements; (2) Zn-coprecipitated ferrihydrite (ZnFh); and (3) Ni-coprecipitated ferrihydrite (NiFh) in both NaHCO3 and HEPES buffer. X-ray diffraction revealed magnetite as the dominant transformation product for all reaction conditions, while siderite was only detected in experiments containing NaHCO3 as buffer. Based on similar initial dissolved Ni and Zn concentrations, we found opposing behavior of Zn and Ni where Zn showed enrichment in abiogenic magnetite, while Ni was more enriched in biogenic magnetite. Combining supposed Zn and Ni concentrations in Precambrian seawater and magnetite mineral grains from three formations deposited at different ages (i.e. 3.75 Ga Nuvvuagittuq Supracrustal Belt, 2.45 Ga Weeli Wolli Iron Formation, and 1.88 Ga Sokoman Iron Formation), we reconstructed potential Zn and Ni partition coefficients between Precambrian seawater and magnetite minerals. The reconstructed potential Zn and Ni partition coefficients suggested that these magnetite minerals from three formations were abiogenic. These differing behaviors suggest the possibility of using Zn and Ni partition coefficient ratios as a means of determining the presence or absence of DIRB in the formation of ancient magnetite deposits.

A 4-methyl-2,6-diformylphenol based fluorescent chemosensor for Al3+

We report here synthesis, characterization and Al3+ ion sensing properties of a 4-methyl-2,6-diformylphenol (DFP) based compound, 4-methyl-2,6-bis-(pyrimidin-2-ylimino)methyl)phenol (HMPM). It has been synthesized by condensation reaction between one eqv. of DFP and two eqv. of 2-aminopyrimidine in acetonitrile under mild conditions. Fluorescence intensity of HMPM increases by 12-fold in the presence of one eqv. of Al3+ in 10 mM HEPES buffer in water:DMSO (1:9, v/v) (pH 7.4). Other cations do not induce any appreciable increment in its fluorescence intensity. Its quantum yield and fluorescence life time enhance upon addition of Al3+. HMPM forms 1:1 complex with the cation. Detection limit has been determined as 1.27 μM. Bare eye detection of Al3+ is possible with the help of HMPM. The probe has been used to detect Al3+ in ‘real sample’.

A dansyl-based fluorescent probe for sensing Cu2+ in aqueous solution

A fluorescent probe based on a glycyl-L-tyrosine-modified dansyl derivative (D-GT) is designed and synthesized. D-GT demonstrated great detection performance toward Cu2+ in an aqueous solution. Fluorescence quenching occurred due to the coordination of Cu2+ with D-GT. The sensitive detection of D-GT to Cu2+ was applied in aqueous solution within a wide pH span (6–12). A 1:1 coordinate stoichiometric way and an association constant of 6.47 × 104 M−1 between D-GT and Cu2+ were determined. The measured detection limit for Cu2+ in HEPES buffer solution (10 mM, pH 7.4) was 0.69 μM. The probe displayed an appropriate sensitivity toward Cu2+ in real drinking water samples and living cells, which reveals the potential applications of D-GT in complicated environments.

A coumarin based visual and fluorometric probe for selective detection of Al(III), Cr(III) and Fe(III) ions through “turn-on” response and its biological application

A Schiff base (L) containing coumarin and pyrene moieties was synthesized and characterized. It showed excellent selective recognition of M(III) ions (M = Al, Cr and Fe) in presence of monovalent, divalent and other trivalent metal ions. Sensor L itself is non-fluorescent upon excitation with 353 nm light (λex = 353) but a very large enhancement in fluorescence intensity was observed during the addition of these three trivalent metal ions into a CH3OH/aqueous HEPES buffer (5 mM, pH = 7.4, 6:4, v/v) solution of L. This prominent “OFF-ON” fluorescence switching can be observed with naked eye, in which “OFF” is due to operation of PET process in free L but PET is inhibited upon complexation thereby allowing CHEF (chelation enhanced fluorescence) process to operate. Detection limits of Al3+, Cr3+ and Fe3+ using L have been found to be 0.79 nM, 1.15 μM, and 1.28 μM respectively in the pH range of 6‒10. Additionally, “OFF-ON” fluorescence can be reversed by sequestering M(III) ions using [EDTA]4– solutions. Analysis of mass spectra and Job’s plot revealed a binding ratio of 1:1 for all three metal ions with L. The imine nitrogen and carbonyl oxygen atoms of L are involved in binding with Al3+, Cr3+ and Fe3+ ions, as revealed by NMR titration and IR spectroscopic analysis. Results of quantum yield (Φ), time resolved photoluminescence (TRPL) and computational studies (DFT/TDDFT) studies were also reported. Fluorescence intensity of these M(III) bound L complexes is quenched by fluoride ions. From cytotoxicity analysis, 7.5 μM of probe L was selected to analyze its fluorescence attributes in cellular imaging. Using human breast cancer cell line (MDA-MB-231) and primary human dermal fibroblasts (HDF), the potentiality of the probe L was confirmed through fluorescence cell imaging experiments.

Engineering a mitochondria-targeted ratiometric fluorescent probe with a large Stokes shift for H2S-specific assaying in foodstuffs and living cells

Hydrogen sulfide (H2S) is a toxic gas found in foods. On-site monitoring hydrogen sulfide level in foodstuffs is desired. Hence, a mitochondria-targeted ratiometric fluorescent sensor (NBP-HS) with a large Stokes shift for H2S detection was constructed based on assembling 2-thiophenecarbonyl group and merocyanine (T-B). The fluorescence ratio (I590/I520) of NBP-HS displayed a good linear relationship related to H2S concentration (0−60 μM) with the detection limit of 5.4 × 10−8 M in HEPES buffer. The sensing mechanism was determined by HRMS analysis and DFT calculation. Interestingly, the H2S-response ratiometric probe was successfully used to on-site measure the freshness of meat by recording the color change. The analysis results of test strips were completely consistent with the analysis results of the colonies count. The probe shows an excellent response to H2S in ratiometric fluorescence in living cells, illustrating the broad applicability of NBP-HS for imaging in biosystems.

Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.

Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC–MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as “Mixing QC.” This Mixing QC enables the detection of TMT labeling issues by LC–MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.

Measurement of Free Testosterone in Serum Using Equilibrium DialysisCoupled With ID-UHPLC-MS/MS: Comparison Between Equilibrium Devices

Free testosterone (FT) has been used as a biomarker in clinical patient care and public health research to assess and manage patients with androgenic abnormalities. The latest Endocrine Society clinical practice guideline for testosterone therapy in men with hypogonadism recommends measuring FT for those with borderline and low total testosterone concentrations, or those who have conditions that change SHBG concentrations, such as some metabolic or hormonal diseases, certain medication use, or SHBG genetic polymorphisms. Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ID-UHPLC-MS/MS).A serum sample was dialyzed against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute M914) was used to prepare calibrators, enabling traceable quantitation and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. The dialysis step requires maintaining the endogenous free hormone equilibrium so that results in dialysate reflect FT concentrations in the blood without influence from dilution, temperature, or pH. The dialyzer system has a 1:1 sample-to-buffer volume and has been used in reference measurement procedures for free hormone measurements, serving as the standard for method performance comparison. Four commercially available devices designed for high throughput in a multiple well-plate format, requiring respective sample-to-buffer ratios, were evaluated for their recovery, speed, ease of automation by a liquid handling system, repeatability, and robustness. Preliminary results showed that a device with 1:1 sample-to-buffer volume had the most comparable results to those obtained from the standard dialyzer, with the mean bias less than 15%. The device with the highest sample-to-buffer ratio showed bias as high as 50%. These data suggest that controlling sample-to-buffer ratio is a critical step in ED FT method.