Spectrophotometric Readout Research Articles

Development and application of global assays of hyper‐ and hypofibrinolysis

Numerous methods for evaluation of global fibrinolytic activity in whole blood or plasma have been proposed, with the majority based on tissue‐type plasminogen activator (t‐PA) addition to initiate fibrinolysis. We propose that such an approach is useful to reveal hypofibrinolysis, but t‐PA concentrations should be kept to a minimum. In this paper, we describe a low‐concentration t‐PA plasma turbidity assay to evaluate several congenital factor deficiencies, including plasminogen activator inhibitor‐1 (PAI‐1) and plasminogen deficiency, as well as hemophilia A and B. In addition, we demonstrate a threshold dependency on endogenous PAI‐1 levels. To assess endogenous hyperfibrinolysis, we suggest that assays that avoid t‐PA addition are preferable, with assays based on euglobulin fractionation remaining a viable choice. We describe a euglobulin fraction clot lysis time (ECLT) assay with spectrophotometric readout and other modifications, and evaluate it as a tool to measure hyperfibrinolysis in inherited clotting factor deficiency states. We demonstrate that the ECLT is predominantly driven by residual amounts of PAI‐1, t‐PA, and α2‐antiplasmin. These assays should be further evaluated for the detection of hypo‐ or hyperfibrinolysis in acquired thrombotic or hemorrhagic disorders.

A molecularly imprinted polymer undergoing a color change depending on the concentration of bisphenol A.

A molecularly imprinted system is introduced here whose color gradually changes as the analyte becomes rebound. The MIP was prepared from an acryloyl-modified β-cyclodextrin (β-CD), acrylamide (AAm), and N,N'-methylenebisacrylamide (MBAA), and imprinted with bisphenol A (BPA). The sensing capability of the MIP was first tested by potentiometry. A spin-coated gold plate coated with the MIP was used as a working electrode; the electrode can differentiate BPA from phenol or p-cresol, which were used as analogs of BPA. Next, a color-responsive system was fabricated by forming a hydrogel membrane containing the modified β-CD, AAm, and MBAA. A vesicle solution was prepared from N-(2-aminoethyl)pentacosa-10,12-diynamide by sonication and incorporated into the hydrogel. The blue polydiacetylene was formed by UV photopolymerization. In the presence of BPA, this system undergoes a color change from blue to red that is proportional to the degree of BPA rebinding. The color change is due to the contraction of the gel membrane that rebinding causes. The method works to 0.5mM BPA concentration range. The detection limits for BPA are 0.1mM on visual assessment and 50μM on spectrophotometric readout. Graphical AbstractA molecular imprinting system is describedwhose color changes from blue to redas it binds bisphenol A. The degree of rebinding can be measured by detecting the color change of polydiacetylene vesicle. CD: cyclodextrin, BPA: bisphenol A.

Gold Nanostar Colorimetric Detection of Fructosyl Valine as a Potential Future Point of Care Biosensor Candidate for Glycated Haemoglobin Detection.

Diabetes Mellitus is a growing global concern. The current methods used to detect glycated haemoglobin are precise, however, utilise expensive equipment, reagents and consumables. These are luxuries which rural communities cannot access. The nanotechnology methods which have been developed for glycated haemoglobin detection are predominantly electrochemically based, have complicated lengthy fabrication processes and utilise toxic chemicals. Here a fructosyl amino acid oxidase gold nanostar biosensor has been developed as a potential future point of care biosensor candidate for glycated haemoglobin detection. The workup done on this biosensor showed that it was able to give a spectrophotometric readout and colorimetric result with naked eye detection in blank serum spiked with fructosyl valine.

The removal of Triton X-100 by dialysis is feasible!

Triton X-100 has been widely used in many analytical and preparative protocols for a long time. Nevertheless, mass spectrometry, chromatographic separation, and spectrophotometric readout may be considerably hampered by this detergent due to signal suppression, complex formation, and high blank values, respectively. Additionally, Triton X-100 is not safe to remove prior to analytics. Here, microdialysis is introduced as a parallelizable, high-throughput method to clean samples from Triton X-100 with high efficacy and precision. To achieve this, we exploit the potential to considerably increase the critical micellar concentration of Triton X-100 by alteration of matrix properties. To that end, addition of several chaotropic compounds and organic solvents has been shown to increase the critical micellar concentration as well as the removal rate of the detergent. For application, matrix additives can be selected for analyte stability requirements out of a variety of compounds. Conveniently, all these additives are removable subsequently using the same microdialysis tool for downstream analytics requirements. Applicability and protocols are shown with proteomic sample preparation of purified proteins and complex protein mixtures prior to matrix-assisted laser desorption ionization (MALDI) mass spectrometry.

Pseudo‐hypoproteinemia in a hyperbilirubinemic dog with immune‐mediated hemolytic anemia

A 10-year-old spayed female Miniature Poodle was presented to the University of Georgia veterinary teaching hospital for evaluation of lethargy, vomiting and anorexia of 4 days' duration. Physical examination, history and a minimum database led to a diagnosis of immune-mediated hemolytic anemia accompanied by marked hyperbilirubinemia. Refractometric protein determination was within the reference interval, whereas the biuret method indicated hypoproteinemia. This discrepancy was attributed to interference of bilirubin and biliverdin with the spectrophotometric read-out of the biuret total protein assay. The albumin concentration, determined by bromcresol green, and refractometric total protein were less affected by this interference.

Temperature effect on the gamma dose response of glycine (spectrophotometric read-out) system

Abstract The glycine (spectrophotometric read-out) dosemeter has a useful dose range of 15–4000 Gy with good reproducibility and post-irradiation stability. It is a potential dosemeter for radiation processing applications, but the dose response of the system may vary with irradiation and measurement temperature. The effect of irradiation and variation in measurement temperature on the gamma dose response of this system was investigated. The gamma dose response was found to be constant for both irradiation and measurement temperatures ranging from 10 to 50 °C, and the maximum variation in response is within ±1%.

Sensitization of glycine (spectrophotometric read-out) dosimetric system using sorbitol

Glycine spectrophotometric read-out systems have a useful dose range of 15–4000 Gy. An attempt was made to sensitize it using sorbitol as a sensitizer. Optimum compositions of aqueous acidic solutions of ferrous ammonium sulphate–xylenol orange (XO), i.e. FX and sorbitol–ferrous ammonium sulphate–xylenol orange, i.e. SFX, for 400 mg of glycine, which gives maximum dosimetric response for any given dose, were established. Molar absorption coefficient values of ferric–XO–glycine complex, i.e. ɛ-values, were determined for glycine system in FX and SFX. These values were found to be 8410 and 15,000 m 2 mol −1 respectively, indicating that an enhancement or sensitivity factor of about 1.78 can be achieved by sorbitol for glycine in SFX. This factor was further confirmed by measuring the gamma dose response of glycine in FX and in SFX for four different doses, viz. 37.8, 75.5, 151 and 302 Gy. It was observed that dose response of glycine in SFX is about 77% more than that of glycine in FX. The maximum variation observed in response of glycine in FX or SFX was found to be within ±1.5%.

Dosimetric evaluation of sucrose and granulated cane sugar in the therapeutic dose range

Granulated cane sugar has been used as a dosimetric material to report dose in high dose accidental irradiations. The purpose of this study was to assess whether clinical dosimetry is also plausible with such a commonly available material. The behavior of cane sugar was explored with respect to therapeutically relevant radiation quantities (dose, dose rate) and qualities (energy, radiation type) as well as under different temperature conditions. The stability of the signal postirradiation was also measured. Absorbed dose was measured by spectrophotometric readout of a ferrous ammonium sulfate xylenol orange (FX)-sugar solution in 10 cm path length cells. A visible color change was produced as a function of dose when the irradiated sugar samples were dissolved in FX solution (10% dilution by mass). A comparison of the optical absorbance spectra and dose response of cane sugar with analytical grade sucrose was done to establish a benchmark standard from which subsequent dosimetry measurements can be validated. The response of the sugar dosimeter read at 590 nm was found to be linear over the dose range of 100-2000 cGy, independent of energy (6-18 MV) and of the average dose rate (100-500 cGy/min). The readout of sugar samples irradiated with mixed photon and electron fields was also shown to be independent of radiation type (photons and electrons). Sugar temperature (20-40 degrees C) during irradiation did not affect dose estimates, making it a promising dosimeter for in vivo dosimetry, particularly in cases where the dosimeter must remain in contact with the patient for an extended period of time. Sugar can be used as an integrating dosimeter, since it exhibits no fractionation effects. Granulated cane sugar is cost effective, safe, soft tissue equivalent, and can be used under various experimental conditions, making it a suitable dosimeter for some radiotherapy applications.

Relative depth dose profile measurement in water phantom for Co-60 teletherapy machine using glycine (spectrophotometric read-out) system

Relative depth dose profile measurement in water phantom for Co-60 teletherapy machine using glycine (spectrophotometric read-out) system

Aspartame tablets—gamma dose response and usability for routine radiation processing dosimetry using spectrophotometry

Aspartame tablets were studied for gamma dose response, using spectrophotometric read-out method. The optimum concentration for ferrous ions was 2 × 10 - 4 mol dm - 3 and xylenol orange with 2.5 × 10 - 1 mol dm - 3 of sulphuric acid for the optimum acidity in FX solution. Wavelength of maximum absorbance is 548 nm. Post-irradiation stability is appreciable i.e. for not less than one month. Dose response is non-linear with third order polynomial fit, in the dose range of 1000–10000 Gy. This system of aspartame was further used for carrying out relative percentage dose profile measurement in Gamma Cell-220. Results obtained were inter-compared with that of a glutamine dosimeter, which showed that maximum difference between the values of aspartame and glutamine systems is within ± 10 % .

A high-throughput screening assay for the carboxyltransferase subunit of acetyl-CoA carboxylase

One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance.

Gamma dose measurement by glycine system using spectrophotometric read-out method

Use of glycine system for gamma ray dosimetry has been investigated by a spectrophotometric read-out method. Accordingly, glycine is irradiated in actual irradiation conditions for different gamma ray dosimetry applications such as dose-rate determination at specified locations in irradiation chamber Panoramic Batch Irradiator (PANBIT), dose evaluation in the product irradiation box of the Food Package Irradiator and relative percentage dose distribution profile in central vertical plane of the irradiation volume of Gamma Cell 220. Results obtained were intercompared with that of a standard such as Fricke or glutamine dosimeter, irradiated under identical conditions. Maximum difference observed between glycine value and Fricke or glutamine value, was ± 6 % .

Dosimetric response studies of glycine–FX solutions

Known amount of irradiated glycine was dissolved in FX solution (i.e. aqueous solution containing ferrous ammonium sulphate, xylenol orange (XO) and sulphuric acid) and its dose response was estimated by using spectrophotometric read-out method. Optimum FX composition was determined for different amounts of glycine. Wavelength of maximum absorbance is 547 nm. Post-irradiation stability period is atleast 1 month. Varying the amount of irradiated glycine (viz. 50, 200 or 400 mg) in optimized FX solution and using the required optical pathlength, a dose range of 15–4000 Gy can be measured with variation of <±2% in dose response.

Threonine-FX dosimeter for food irradiation

A detailed study for the spectrophotometric readout method for L-threonine powder, [CH 3CH(OH)CH(NH 2)COOH], was done. In this method, 400 mg unirradiated/irradiated L-threonine powder was dissolved in 10 ml of a solution which contains 3×10 −4 mol dm −3 ferrous ammonium sulphate and 1.7×10 −4 mol dm −3 xylenol orange (XO) in aerated aqueous 0.17 mol dm −3 sulphuric acid (FX). The peroxy radicals produced from irradiated threonine oxidize ferrous ions and XO forms a complex with ferric ions as well as controls the chain length of ferrous ion oxidation. The plot of absorbance at 556 nm against dose is linear in the dose range 20–400 Gy and doses down to about 1 Gy can be measured using 10-cm path cells. Response of the dosimeter is independent of irradiation temperature above 20. A dose of 50 Gy–10 kGy can be measured dissolving 50 mg threonine powder in 10 ml of a solution which contains 3×10 −4 mol dm −3 ferrous ammonium sulphate and 1.3×10 −4 mol dm −3 XO in aerated aqueous 0.06 mol dm −3 sulphuric acid (FX). The plot of absorbance at 552 nm against dose is non-linear. However dosimeter shows linear dose response up to 1000 Gy. Irradiated threonine powder is stable for about 3 months. The reproducibility of the method is better than ±2%. This dosimeter is very useful as transfer dosimeter for food irradiation programme.

Spectrophotometric readout for an alanine dosimeter for food irradiation applications

The alanine–electron spin resonance (EPR) readout system is well known as a reference and transfer dosimetry system for the evaluation of high doses in radiation processing. The high cost of an EPR/alanine dosimetry system is a serious handicap for large-scale routine application in irradiation facilities. In this study, the use of a complex produced by dissolving irradiated l-alanine in 1,4-phenyl diammonium dichloride solution was investigated for dosimetry purposes. This complex—having a purple colour—has an increasing absorbance with increasing dose in the range of 1–20 kGy. The applicability of spectrophotometric evaluation was studied by measuring the absorbance intensity of this complex at 360 and 505 nm, respectively. Fluorimetric evaluation was also investigated by measuring the emission of the complex at 435 nm as a function of dose. The present method is easy for routine application. The effect of the dye concentration as well as the suitable amount of irradiated alanine has been studied. With respect to routine application, the stability of the product complex after its formation was also investigated.

Dose intercomparison for 60Co, 10 MeV electrons and bremsstrahlung radiation using glutamine (spectrophotometric readout) dosimeter at different irradiation temperatures

This paper gives the results of dose intercomparison for 60Co gamma, 10 MeV electron and bremsstrahlung radiation using glutamine (spectrophotometric readout) dosimeter. Both gamma and electron irradiations were done at Riso National Laboratory, Denmark. Gamma irradiation was done at IAEA laboratory, Vienna and bremsstrahlung irradiation was done as part of IAEA CRP programme. The irradiation temperature during these experiments varied from 15 to 43°C and corrections were therefore applied for the irradiation temperatures. These intercomparisons give acceptable results using gamma calibration, which indicate that gamma calibration is valid also for other types of radiation.

Chemical dosimetry techniques for various applications under different geometries

This paper gives the results of dosimetry for various applications under different geometrical arrangements. These applications include: gamma chambers, blood irradiators, radiotherapy using both 60 Co and accelerators, animal irradiations with different types of radiation sources, fluid irradiators for sludge and rubber latex and industrial electron irradiators. The dosimeters used were Fricke, FBX and alanine/glutamine (spectrophotometric readout).

A new spectrophotometric readout for the alanine-triphenyl tetrazolium chloride system for high-dose dosimetry

The electron spin resonance (ESR) readout of radical concentration in irradiated L -alanine is well known as a transfer dosimetry system, however, the high cost of the ESR equipment is a serious handicap for large-scale routine application. In this study the reaction between irradiated L -alanine powder and aqueous solution of triphenyl tetrazolium chloride (TTC) was studied. The radical produced by irradiation of alanine reacts during the dissolution with TTC forming pink-colored formazans with absorption maxima around 401 and 487 nm. This readout method can be used in the 2–200 kGy dose range.

Dose intercomparison between BARC (India) and NPL (UK) using glutamine (spectrophotometric readout) and dichromate dosimeters

Abstract A dose intercomparison was carried out for 60Co gamma ray irradiations between BARC (India) and NPL (UK) using mailed high-dose dosimeters. Both the laboratories maintain standards for radiation processing dosimetry. Glutamine (spectrophotometric readout) dosimeters supplied by BARC were irradiated at NPL and read at BARC. Dichromate dosimeters supplied by NPL were irradiated at BARC and read at NPL. For the glutamine dosimeter, the BARC/NPL dose ratio was 0.997±0.010 (1σ) while for the dichromate dosimeter, the BARC/NPL dose ratio was 0.983±0.006 (1σ). These results show agreement within the combined uncertainties quoted by the two laboratories.

Effect of impurities in xylenol orange on the spectrophotometric readout method for alanine dosimetry

A useful spectrophotometric readout method for alanine dosimetry involves the oxidation of ferrous ions when irradiated alanine powder is dissolved in an acidic aqueous aerated solution (FX) containing ferrous sulphate and xylenol orange (XO). The present investigation shows that the impurities in reagent grade XO affect the ferric-XO-alanine complex formation as well as the characteristic ferric ion concentration vs dose plot. Therefore, a calibration is necessary for any batch of XO used in FX dosimeter solution preparation.