A robust method for simultaneous determination of eight B vitamins in human serum by liquid chromatography tandem mass spectrometry.

The level of vitamin B group in human serum is an important index of human health. Among B vitamins, cyanocobalamin in serum is unstable and its content is extremely low. Rapid and simultaneous detection of multiple B vitamins including cyanocobalamin is a challenge. Herein, we have developed a rapid and stable method that can realize the determination of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin simultaneously in 6 min. The method was established based on protein precipitation with methanol and then chromatographic separation was achieved using Waters acquity ultra-high-performance liquid chromatography high strength silica T3 column, which was stable and sensitive especially for cyanocobalamin. Limit of quantification, precision, trueness, and matrix effect were validated according to the European Medicines Agency and United States Food and Drug guidelines and Clinical and Laboratory Standards Institute guidelines on bioanalytical method. The limit of quantification for thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin was 0.4, 0.4, 0.8, 2.0, 0.4, 0.1, 0.4, and 0.04 ng/mL separately, respectively. Intra- and interday precisions were 1.1%-12.4% and 2.0%-13.5%, respectively. The relative errors were between 0.3% and 13.3%, and the matrix effects were between 2.6% and 10.4%.

Investigation of some quality properties of yogurt made from cow and sheep milk fortified with folic acid (B9 ), biotin (B7 ), and vitamin D3.

BACKGROUND: The aim of this study was to investigate the effects on some physicochemical properties and starter cultures of yogurts enriched with vitamins at different concentrations during storage. For this purpose, yogurt was produced by adding the vitamins folic acid (B9 ), biotin (B7 ), and vitamin D3 in different concentrations to sheep and cow milk and stored at 4 °C. Physicochemical analyses and microbiological analyses were performed for each group of yogurt on days 0, 7, 14, and 21. RESULTS: There was no significant difference (P > 0.05) between the groups in pH and titration acidity (%) during storage. It was determined that in the yogurts produced from sheep milk, the groups enriched with vitamins had a higher number of L. bulgaricus than the control group on the 7th day of storage. Moreover, the groups containing vitamin D3 exhibited a higher Lactobacillus bulgaricus count on the 21st day of storage. The highest L. bulgaricus counts on the 7th day in yogurts produced from cow's milk were observed in groups containing 0.5 mL of vitamin B9 and B7 . No mold or yeast growth was observed during storage in any of the yogurt groups made from cow and sheep milk. CONCLUSION: In conclusion, it was determined that the enrichment of yogurt with vitamins B7 , B9 , and D3 did not adversely affect the quality of the yogurt; rather, it improved. We recommend that yogurt enriched with micronutrients be studied economically, and mass production should be initiated by yogurt companies as soon as possible. © 2023 Society of Chemical Industry.

Magnetic alignment of rhodamine/magnetite dual-labeled microtubules probed with inverted fluorescence microscopy.

Molecular machines, as exemplified by the kinesin and microtubule system, are responsible for molecular transport in cells. The monitoring of the cellular machinery has attracted much attention in recent years, requiring sophisticated techniques such as optical tweezers, and dark field hyperspectral and fluorescence microscopies. It also demands suitable procedures for immobilization and labeling with functional agents such as dyes, plasmonic nanoparticles and quantum dots. In this work, microtubules were co-polymerized by incubating a tubulin mix consisting of 7 biotinylated tubulin to 3 rhodamine tubulin. Rhodamine provided the fluorescent tag, while biotin was the anchoring group for receiving streptavidin containing species. To control the microtubule alignment and consequently, the molecular gliding directions, functionalized iron oxide nanoparticles were employed in the presence of an external magnet field. Such iron oxide nanoparticles, (MagNPs) were previously coated with silica and (3-aminopro-pyl)triethoxysilane (APTS) and then modified with streptavidin (SA) for linking to the biotin-functionalized microtubules. In this way, the binding has been successfully performed, and the magnetic alignment probed by Inverted Fluorescence Microscopy. The proposed strategy has proved promising, as tested with one of the most important biological structures of the cellular machinery.

Design of novel tripyridinophane-based Eu(III) complexes as efficient luminescent labels for bioassay applications.

In this work, the development of highly luminescent europium(III) complexes in water solution is reported, including their syntheses, analyses of their photophysical properties and applications in bioassays. Three Eu(III) complexes are derived from new ligands based on a tripyridinophane platform. There are four distinct sections in the structure of these ligands: an 18-membered polyaminocarboxylic macrocycle to bind efficiently lanthanide ions in aqueous solutions, three chromophoric subunits (4-(phenylethynyl)pyridine moieties) to effectively sensitize the emission of the metal, two peripheral moieties to solubilise the complex in aqueous media (sulfonate, sulfobetaine or glucose groups) and a free NH2 group available for grafting or bioconjugation. In our synthetic procedure, a pivotal macrocyclic platform is obtained with a high yield in the crucial macrocyclization step due to a metal template ion effect (74% yield). In Tris aqueous buffer (pH 7.4), the Eu(III) complexes show a maximum excitation wavelength at 320 nm, a suitable overall quantum yield (14%), a relatively long lifetime (0.80 ms) and a one-photon brightness in the range of 10 000 M-1 cm-1. Importantly, these photophysical properties are retained at dilute concentrations, even in the presence of a very large excess of potentially competing species, such as EDTA or Mg2+ ions. Furthermore, we report the bioconjugation of a Eu(III) complex labelled by an N-hydroxysuccinimide ester reactive group with an antibody (anti-glutathione-S-transferase) and the successful application of the corresponding antibody conjugate in the detection of GST-biotin in a fluoroimmunoassay. These new complexes provide a solution for high sensitivity in Homogeneous Time-Resolved Fluorescence (HTRF®) bioassays.

Biotin as a masking agent in chorionic gonadotropin assays utilizing biotinylated antibodies.

Biotin interference in streptavidin/biotin-based immunoassays has been recently recognized as a confounding factor in clinical settings. Depending on the nature of the assay, the presence of excess biotin in patient samples can cause falsely high or low results. One of the platforms known to be affected, Roche Cobas, is widely used in anti-doping laboratories to test for intact chorionic gonadotropin (hCG) in urine. While biotin levels in blood have been well studied, less is known about urinary biotin due to its limited clinical significance. Having analyzed over 4,000 urine samples, we have established a reference range for urinary biotin with a median concentration of approximately 12 ng/ml. However, a significant number of samples contain much higher amounts, with a maximum approaching 10 µg/ml, suggesting biotin supplementation. Consequently, the tolerance of hCG STAT assay towards biotin was investigated over a wide concentration range. The apparent hCG concentration was found to decrease almost linearly as biotin increased from 100 to 1,000 ng/ml, with only 10% of the expected value reported by the assay as biotin reached 1,000 ng/ml. Further increase of biotin resulted in a progressive, albeit more moderate, decline in measured hCG concentration. To avoid a false negative result in the context of anti-doping analysis, it is highly recommended to monitor biotin in urine and perform diafiltration before hCG measurement in samples with elevated biotin to remove the interference.

Biotin Interference in Assays for Thyroid Hormones, Thyrotropin and Thyroglobulin.

Background: Biotin has been reported to interfere with several commonly used laboratory assays resulting in misleading values and possible erroneous diagnosis and treatment. This report describes a prospective study of possible biotin interference in thyroid-related laboratory assays, with a comparison of different commonly used assay platforms. Materials and Methods: Thirteen adult subjects (mean age 45 ± 13 years old) were administered biotin 10 mg/day for eight days. Blood specimens were collected at three time points on day 1 and on day 8 (baseline, two, and five hours after biotin ingestion). Thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), total triiodothyronine (TT3), total thyroxine (TT4), thyroxine binding globulin (TBG), and thyroglobulin (Tg) levels were analyzed with four different platforms: Abbott Architect, Roche Cobas 6000, Siemens IMMULITE 2000, and liquid chromatography with tandem mass spectrometry (LC-MS/MS). TSH, fT3, fT4, TT3, and TT4 were measured with Abbott Architect and Roche Cobas 6000. fT3, fT4, TT3, and TT4 were also measured by LC-MS/MS. Tg was measured by Siemens IMMULITE 2000. TBG was assessed with Siemens IMMULITE 2000. Results: Significant changes in TSH, fT4, and TT3 measurements were observed after biotin exposure when the Roche Cobas 6000 platform was used. Biotin intake resulted in a falsely lower Tg level when measurements were performed with Siemens IMMULITE 2000. At the time points examined, maximal biotin interference was observed two hours after biotin exposure both on day 1 and day 8. Conclusions: A daily dose of 10 mg was shown to interfere with specific assays for TSH, fT4, TT3, and Tg. Physicians must be aware of the potential risk of erroneous test results in subjects taking biotin supplements. Altered test results for TSH and Tg can be particularly problematic in patients requiring careful titration of levothyroxine therapy such as those with thyroid cancer.

Human platelets labeled at two discrete biotin densities are functional in vitro and are detected in vivo in the murine circulation: A promising approach to monitor platelet survival in vivo in clinical research.

BACKGROUND: The production of platelet concentrates (PCs) is evolving, and their survival capacity needs in vivo evaluation. This requires that the transfused platelets (PLTs) be distinguished from those of the recipient. Labeling at various biotin (Bio) densities allows one to concurrently trace multiple PLT populations, as reported for red blood cells. STUDY DESIGN AND METHODS: A method is described to label human PLTs at two densities of Bio for future clinical trials. Injectable-grade PLTs were prepared in a sterile environment, using injectable-grade buffers and good manufacturing practices (GMP)-grade Sulfo-NHS-Biotin. Sulfo-NHS-Biotin concentrations were chosen to maintain PLT integrity and avoid potential alloimmunization while enabling the detection of circulating BioPLTs. The impact of biotinylation on human PLT recirculation was evaluated in vivo in a severe immunodeficient mouse model using ex vivo flow cytometry. RESULTS: BioPLTs labeled with 1.2 or 10 µg/ml Sulfo-NHS-Biotin displayed normal ultrastructure and retained aggregation and secretion capacity and normal expression of the main surface glycoproteins. The procedure avoided detrimental PLT activation or apoptosis signals. Transfused human BioPLT populations could be distinguished from one another and from unlabeled circulating mouse PLTs, and their survival was comparable to that of unlabeled human PLTs in the mouse model. CONCLUSIONS: Provided low Sulfo-NHS-Biotin concentrations (<10 µg/ml) are used, injectable-grade BioPLTs comply with safety regulations, conserve PLT integrity, and permit accurate in vivo detection. This alternative to radioisotopes, which allows one to follow different PLT populations in the same recipient, should be valuable when assessing new PC preparations and monitoring PLT survival in clinical research.

Detection of biotin with zeptomole sensitivity using recombinant spores and a competition assay.

Detection of protein-binding analytes is important for many applications. Currently, various instrument-based techniques are used for detecting protein-binding analytes. However, such techniques have several limitations including high cost and time-consuming sample processing. In order to overcome these limitations, we developed a sensitive competition assay for the detection of protein-binding analytes using recombinant endospores as a sensing element. The method is based on the competition between the biotin, the model analyte, and a biotin-magnetic bead complex to bind the recombinant spores containing the biotin binding region of streptavidin. After magnetic attraction, the residual spores in the suspension are spread on plates to form colonies which are used to count the amount of the residual spores; the higher the residual ratio of spores, the more biotin in the samples. The linear range was from 150 zmol to 1.5 fmol and the limit of detection of the assay was 150 zmol. The assay proposed herein is sensitive and does not require any expensive equipment. It is suitable for qualitative or semi-quantitative analysis such as screening tests for the detection of toxic chemicals.

Design of a Multiuse Photoreactor To Enable Visible-Light Photocatalytic Chemical Transformations and Labeling in Live Cells.

Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and in vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.

Balanced Detection Method Using Optical Affinity Sensors for Quick Measurement of Biomolecule Concentrations.

To determine the concentration of biomolecules using a label-free optical biosensor, it is necessary to measure the serial signal from the reaction starting point, which is inconvenient for practical applications. Here, we propose an alternative detection method for determining the concentration of a biomolecule. The method, which is derived from the fraction bound equation of the Langmuir adsorption model, determines the concentration relative to a reference sample with required accuracy, with a single measurement at any point in time. We also experimentally demonstrated the method and its accuracy by detecting streptavidin-biotin complexes using on-chip optical sensors based on active disk resonators integrated with microfluidic circuits. By performing the proposed method in a simultaneous parallel measurement scheme, signal fluctuations evenly induced in the detectors by external perturbations could be automatically suppressed, similar to the balanced detection method. We expect our approach to be applicable to practical applications where fast and accurate detection responses are needed.

Silver nanosol SERS quantitative analysis of ultratrace biotin coupled N-doped carbon dots catalytic amplification with affinity reaction.

Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm-1. With the increase of nancatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006-0.021 ng/mL, with a detection limit of 0.3 pg/mL.

Mitigating biotin interference in two Roche immunoassays by premixing biotinylated capturing molecules with streptavidin coated beads.

BACKGROUND: Biotin is an interference in many streptavidin-biotin based immunoassays, causing falsely decreased results with sandwich immunoassays and falsely increased results with competitive immunoassays. It has been discussed that premixing streptavidin coated beads and biotinylated capturing molecules may prevent biotin interference. This study was designed to test whether such modification could mitigate biotin interference in two originally susceptible sandwich immunoassays. METHODS: Roche C-peptide and human growth hormone (hGH) immunoassays utilize three reagent containers for streptavidin coated beads (M), biotinylated capturing antibody (R1) and ruthenylated antibody (R2). The reagents were modified by premixing reagent M and R1. Following incubation, the beads were placed back in the M-container and R1-supernatant back to R1-container. Patient specimens were selected, spiked with biotin to 1055 ng/mL, and measured by both the original, unmodified reagent and modified reagent on Roche cobas e411 analyzer. The biotin interference dose response curves were also compared using pooled patient specimen spiked with different concentrations of biotin. RESULTS: For the original reagent, 1055 ng/mL of biotin decreased C- peptide results by 88% and hGH results by 97%. After reagent modification, this interference effect was nearly eliminated for C- peptide but remained about 20% decreased for hGH. CONCLUSION: Premixing streptavidin beads and biotinylated capturing molecules is an effective approach to mitigate biotin interference for certain immunoassays.

Signature Fragment Ions of Biotinylated Peptides.

The use of biotin or biotin-containing reagents is an essential component of many protein purification and labeling technologies. Owing to its small size and high affinity to the avidin family of proteins, biotin is a versatile molecular handle that permits both enrichment and purity that is not easily achieved by other reagents. Traditionally, the use of biotinylation to enrich for proteins has not required the detection of the site of biotinylation. However, newer technologies for discovery of protein-protein interactions, such as APEX and BioID, as well as some of the click chemistry-based labeling approaches have underscored the importance of determining the exact residue that is modified by biotin. Anti-biotin antibody-based enrichment of biotinylated peptides (e.g., BioSITe) coupled to LC-MS/MS permit large-scale detection and localization of sites of biotinylation. As with any chemical modification of peptides, understanding the fragmentation patterns that result from biotin modification is essential to improving its detection by LC-MS/MS. Tandem mass spectra of biotinylated peptides has not yet been studied systematically. Here, we describe the various signature fragment ions generated with collision-induced dissociation of biotinylated peptides. We focused on biotin adducts attached to peptides generated by BioID and APEX experiments, including biotin, isotopically heavy biotin, and biotin-XX-phenol, a nonpermeable variant of biotin-phenol. We also highlight how the detection of biotinylated peptides in high-throughput studies poses certain computational challenges for accurate quantitation which need to be addressed. Our findings about signature fragment ions of biotinylated peptides should be helpful in the confirmation of biotinylation sites.

Preservation of vitamins content in Cuccìa using an innovative method of processing.

Cuccìa is a traditional Sicilian food prepared by boiling whole durum wheat kernels, in water, for many hours. This process destroys the vitamins E and B contents of crude kernels. It was rated a method to prepare the Cuccìa, preserving the vitamin content. Four varieties of durum wheat were processed comparing the traditional cooking method (TR-boiling for 5/6 hours), and an innovative one (IN-grains scarification, germination, and cooking at 50 °C for 2 hours). On soups obtained the content of biotin, niacin and α-amylase activity were determined. ANOVA showed the cooking method influences biotin and niacin content having values from 0.56 and 0.72 ng ml-1 (raw grain) and values close to 0 (TR), while only a 10% decrease (IN) respectively for both vitamins. On the contrary, α-amylase activity was reduced with IN method. The IN method combined with ancient grains, produces the soup with a good vitamin B amount.

Electrothermal Active Control of Preconcentrated Biomolecule Plugs.

Concentration-polarization (CP)-based biomolecule preconcentration is highly effective in enhancing the detection sensitivity yet fails to precisely and dynamically control the location of the preconcentrated biomolecule plug to ensure overlap with the sensing region (e.g., immobilized molecular probes). Here, we used electrothermal (ET) stirring as a means of controlling the location of a preconcentrated biomolecule plug. The applied microfluidic device consisted of a Nafion membrane to induce the CP and an array of individually addressable microscale heaters for active local ET stirring. The experimental results demonstrated that such a novel platform enabled active control of the location of the preconcentrated plug of target biomolecules, ensuring its overlap with the functionalized microparticles and ultimately yielding enhanced detection sensitivity and binding kinetics. This was demonstrated using avidin-biotin particles as a simple bead-based bioassay model.

QTR-FRET: Efficient background reduction technology in time-resolved förster resonance energy transfer assays.

A novel homogeneous assay system QTR-FRET (Quencher modulated Time-Resolved Förster Resonance Energy Transfer) combining quenching resonance energy transfer (QRET) and time-resolved Förster resonance energy transfer (TR-FRET) was developed to reduce background signal in the conventional energy transfer applications. The TR-FRET functionality is often limited by the lanthanide donor background signal leading to the use of low donor concentration. QTR-FRET reduces this background by introducing soluble quencher molecule, and in this work the concept functionality was proven and compared to previously introduced QRET and TR-FRET technologies. Comparison was performed with three different Eu3+-chelates exhibiting different luminescent lifetime and stability. The side-by-side comparison of the three signaling systems and Eu3+-chelates was demonstrated in a model assay with Eu3+-chelate conjugated biotin and streptavidin (SA) or Cy5-SA conjugate. Comparison of the methodologies showed increased signal-to-background ratios when comparing QTR-FRET to TR-FRET, especially at high Eu3+-biotin concentrations. Quenching the non-bound Eu3+-biotin improved the assay performance, which suggests that an improved assay performance can be attained with the QTR-FRET method. QTR-FRET is expected to be especially useful for Eu3+-labeled ligands with low affinity or assays requiring high Eu3+-ligand concentration. The QTR-FRET indicated potential for multi-analyte approaches separately utilizing the direct QRET-type Eu3+-chelate signal and energy transfer signal readout in a single-well. This potential was hypothesized with Avi-KRAS nucleotide exchange assay as a second biologically relevant model system.

Biotinylation of platelets for transfusion purposes a novel method to label platelets in a closed system.

BACKGROUND: Labeling of platelets (PLTs) is required to measure the recovery and survival of transfused PLTs in vivo. Currently a radioactive method is used to label PLTs. However, application of those radiolabeling methods is limited by both safety issues and the inability to isolate transfused PLTs from the circulation. Biotin-labeled PLTs are an attractive nonradioactive option. However, no validated protocol to biotinylate PLTs is currently available for human studies. STUDY DESIGN AND METHODS: Six PLT concentrates (PCs) were subaliquoted and biotinylated on Days 1 and 7 of storage. To distinguish the effect of the processing steps from the effects of biotin incubation, two control groups were used: 1) "sham" samples were processed without the biotinylation reagent and 2) control samples were assessed without any processing other than the PC isolation. For the biotinylation procedure, 50 mL of PCs was washed twice and incubated with 5 mg/L biotin for 30 minutes in a closed system. As measures of PLT activation, phosphatidylserine exposure and CD62p expression were assessed. RESULTS: After biotinylation, 98.4% ± 0.9% of PLTs were labeled. PLT counts, pH, and "swirling" were within the range accepted by the Dutch blood bank for standard PLT products. Biotinylated PLTs were more activated compared than controles but not more than sham samples, but were more activated than the controls. CONCLUSION: We developed a standardized and reproducible protocol according to Good Practice Guidelines standards, for biotin labeling of PLTs for clinical purposes. This method can be applied as nonradioactive alternative assess survival and recovery of transfused PLTs in vivo.

A Nucleic Acid Nanostructure Built through On-Electrode Ligation for Electrochemical Detection of a Broad Range of Analytes.

For an assay to be most effective in point-of-care clinical analysis, it needs to be economical, simple, generalizable, and free from tedious workflows. While electrochemistry-based DNA sensors reduce instrumental costs and eliminate complicated procedures, there remains a need to address probe costs and generalizability, as numerous probes with multiple conjugations are needed to quantify a wide range of biomarkers. In this work, we have opened a route to circumvent complicated multiconjugation schemes using enzyme-catalyzed probe construction directly on the surface of the electrode. With this, we have created a versatile DNA nanostructure probe and validated its effectiveness by quantification of proteins (streptavidin, anti-digoxigenin, anti-tacrolimus) and small molecules (biotin, digoxigenin, tacrolimus) using the same platform. Tacrolimus, a widely prescribed immunosuppressant drug for organ transplant patients, was directly quantified with electrochemistry for the first time, with the assay range matching the therapeutic index range. Finally, the stability and sensitivity of the probe was confirmed in a background of minimally diluted human serum.