Stopped-flow Mixing Technique Research Articles

Rapid mixing of colliding picoliter liquid droplets delivered through-space from piezoelectric-actuated pipettes characterized by time-resolved fluorescence monitoring.

Rapid mixing of aqueous solutions is a crucial first step to study the kinetics of fast biochemical reactions with high temporal resolution. Remarkable progress toward this goal has been made through the development of advanced stopped-flow mixing techniques resulting in reduced dead times, and thereby extending reaction monitoring capabilities to numerous biochemical systems. Concurrently, piezoelectric actuators for through-space liquid droplet sample delivery have also been applied in several experimental systems, providing discrete picoliter sample volume delivery and precision sample deposition onto a surface, free of confinement within microfluidic devices, tubing, or other physical constraints. Here, we characterize the inertial mixing kinetics of two aqueous droplets (130 pl) produced by piezoelectric-actuated pipettes, following droplet collision in free space and deposition on a surface in a proof of principle experiment. A time-resolved fluorescence system was developed to monitor the mixing and fluorescence quenching of 5-carboxytetramethylrhodamine (5-Tamra) and N-Bromosuccinimide, which we show to occur in less than 10 ms. In this respect, this methodology is unique in that it offers millisecond mixing capabilities for very small quantities of discrete sample volumes. Furthermore, the use of discrete droplets for sample delivery and mixing in free space provides potential advantages, including the elimination of the requirement for a physical construction as with microfluidic systems, and thereby makes possible and extends the experimental capabilities of many systems.

Kinetic Pathway of the Cylinder-to-Sphere Transition in Block Copolymer Micelles Observed in Situ by Time-Resolved Neutron and Synchrotron Scattering.

Here we present an in situ study of the nonequilibrium cylinder-to-sphere morphological transition kinetics on the millisecond range in a model block copolymer micelle system revealing the underlying mechanism and pathways of the process. By employing the stopped-flow mixing technique, the system was rapidly brought (≈100 μs) deep into the instability region, and the kinetics was followed on the time scale of milliseconds using both time-resolved small-angle neutron and X-ray scattering (TR-SANS and TR-SAXS, respectively). Due to the difference in contrast and resolution, SAXS and SANS provide unique complementary information. Our analysis shows that the morphological transition is characterized by a single rate constant indicating a two-state model where the transition proceeds through direct decomposition (fragmentation) of the cylinders without any transient intermediate structures. The cylindrical segments formed in the disintegration process subsequently grow into spherical micelles possibly through the molecular exchange mechanism until near equilibrium micelles are formed. The observation of a two-step kinetic mechanism, fluctuation-induced fragmentation and ″ripening″ processes, provides unique insight into the nonequilibrium behavior of block copolymer micelles in dilute solutions.

Direct Observation of the Formation of Surfactant Micelles under Nonisothermal Conditions by Synchrotron SAXS

Self-assembly of amphiphilic molecules into micelles occurs on very short times scales of typically some milliseconds, and the structural evolution is therefore very challenging to observe experimentally. While rate constants of surfactant micelle kinetics have been accessed by spectroscopic techniques for decades, so far no experiments providing detailed information on the structural evolution of surfactant micelles during their formation process have been reported. In this work we show that by applying synchrotron small-angle X-ray scattering (SAXS) in combination with the stopped-flow mixing technique, the entire micelle formation process from single surfactants to equilibrium micelles can be followed in situ. Using a sugar-based surfactant system of dodecyl maltoside (DDM) in dimethylformamide (DMF), micelle formation can be induced simply by adding water, and this can be followed in situ by SAXS. Mixing of water and DMF is an exothermic process where the micelle formation process occurs under nonisothermal conditions with a temperature gradient relaxing from about 40 to 20 °C. A kinetic nucleation and growth mechanism model describing micelle formation by insertion/expulsion of single molecules under nonisothermal conditions was developed and shown to describe the data very well.

The Kinetics and Folding Pathways of Intramolecular G-Quadruplex Nucleic Acids

The folding kinetics of G-quadruplex forming sequences is critical to their capacity to influence biological function. While G-quadruplex structure and stability have been relatively well studied, little is known about the kinetics of their folding. We employed a stopped-flow mixing technique to systematically investigate the potassium-dependent folding kinetics of telomeric RNA and DNA G-quadruplexes and RNA G-quadruplexes containing only two G-quartets formed from sequences r[(GGA)(3)GG] and r[(GGUUA)(3)GG]. Our findings suggest a folding mechanism that involves two kinetic steps with initial binding of a single K(+), irrespective of the number of G-quartets involved or whether the G-quadruplex is formed from RNA or DNA. The folding rates for telomeric RNA and DNA G-quadruplexes are comparable at near physiological [K(+)] (90 mM) (τ = ~60 ms). The folding of a 2-quartet RNA G-quadruplex with single nucleotide A loops is considerably slower (τ = ~700 ms), and we found that the time required to fold a UUA looped variant (τ > 100 s, 500 mM K(+)) exceeds the lifetimes of some regulatory RNAs. We discuss the implications of these findings with respect to the fundamental properties of G-quadruplexes and their potential functions in biology.

Determination of antioxidant additives in foodstuffs by direct measurement of gold nanoparticle formation using resonance light scattering detection

The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles has been kinetically studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique and resonance light scattering as detection system. This study has given rise to a simple and rapid method for the determination of several synthetic and natural antioxidants used as additives in foodstuff samples. The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about 5 s, using an integration time of 0.1 s. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of the analytes, were (μmol L −1): gallic acid (0.04–0.59, 0.01), propyl gallate (0.04–1.41, 0.01), octyl gallate (0.03–0.35, 0.08), dodecyl gallate (0.02–0.30, 0.007), butylated hydroxyanisol (0.07–0.39, 0.009), butylated hydroxytoluene (0.04–0.32, 0.01), ascorbic acid (0.11–1.72, 0.03) and sodium citrate (0.07–1.29, 0.02). The regression coefficients were higher than 0.994 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels of each analyte, with values ranging between 0.6 and 4.8%. The practical usefulness of the developed method was demonstrated by the determination of several antioxidant additives in foodstuff samples, which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5%. The results obtained were validated using two reference methods.

Simultaneous determination of captopril and hydrochlorothiazide by time-resolved chemiluminescence with artificial neural network calibration

The combined use of chemometrics and chemiluminescence (CL) measurements, with the aid of the stopped-flow mixing technique, developed a simple time-resolved CL method for the simultaneous determination of captopril (CPL) and hydrochlorothiazide (HCT). The stopped-flow technique in a continuous-flow system was employed in this work in order to emphasize the kinetic differences between the two analytes in cerium (IV)-rhodamine 6G CL system. After the flow was stopped, an initial rise of CL signal was observed for HCT standards, while a direct decay of CL signal was obtained for CPL standards. The mixed CL signal was monitored and recorded on the whole process of continuousflow/stopped-flow, and the obtained data were processed by the chemometric approach of artificial neural network. The relative prediction error (RPE) of CPL and HCT was 5.9% and 8.7%, respectively. The recoveries of CPL and HCT in tablets were found to fall in the range between 95% and 106%. The proposed method was successfully applied to the simultaneous determination of CPL and HCT in a compound pharmaceutical formulation.

Determination of N-acetylcysteine via its effect on the aggregation of gold nanoparticles

The effect of thiol compounds on the kinetics of the aggregation of gold nanoparticles in the presence of the cationic surfactant cetyltrimethyl ammonium bromide has been studied. It was applied to the determination of N-acetylcysteine using the stopped-flow mixing technique along with light scattering detection. The signal obtained was measured after about 5 s, and gave the analytical information for a calibration graph in the concentration range from 2.9 to 60 μmol L−1 of N-acetylcysteine, and a detection limit of 0.87 μmol L−1. The effect of other thiols on the system is also described. The relative standard deviation ranges between 0.6% and 3.5%. The method was applied to the determination of N-acetylcysteine in several pharmaceutical samples with recoveries that range from 97.7% to 101.1%.

Long-wavelength homogeneous enzyme immunoassay for the determination of amikacin in water samples

A simple and rapid homogeneous enzyme immunoassay involving the use of the malic dehydrogenase enzyme and a long-wavelength fluorophor, the oxazine Cresyl Violet, is proposed for the determination of the antibiotic amikacin in water samples. An enzymatic tracer has been synthesized by covalent binding of amikacin to malic dehydrogenase via a carbodiimide derivative. Free tracer catalyses the reaction between Cresyl Violet and malic acid giving rise to a decrease in the fluorescence of the fluorophor. Kinetic curves for this reaction have been monitored at λ ex 585 and λ em 624 nm using the stopped-flow mixing technique, being the initial rate measured in only 2–3 s. The dynamic range of the method is 1–15 ng mL −1 and the detection limit is 0.3 ng mL −1, using aqueous standard solutions or water samples. The precision, obtained at 1 and 5 ng mL −1 and expressed as relative standard deviation, was 6.0 and 9.6%, respectively. The method has been applied to the analysis of drinking, river and wastewater samples. The sample pre-treatment involved a solid-phase extraction step for the clean-up of the samples. A recovery study was carried out to validate the method, being the values obtained in the range 80–114%, with a mean value of 96.7%.

Stopped-flow kinetic method for the fluorimetric determination of DNA traces in biological samples based on the interaction long-wavelength fluorophor, surfactant and nucleic acid

A simple and rapid method for the determination of DNA, involving the interaction between a surfactant, a long-wavelength fluorophor (LWF) and the nucleic acid, is presented. Different chemical systems based on the local effective charge of the surfactant/LWF system with DNA were tested, choosing cetyltrimethyl ammonium bromide (CTAB) and indocyanine green (ICG) for the development of the method. The fluorescence of ICG increases in the presence of CTAB, but it rapidly decreases in the presence of deoxyribonucleic acid. The initial reaction-rate ( v 0 ) and signal at a prefixed-time (Δ IF 20) are monitored at 780 and 802 nm as excitation and emission wavelengths, respectively, using stopped-flow mixing technique, which makes the method applicable to automate routine analysis. Each measurement was obtained in about 30 s, being the integration time 0.1 s. The dynamic range of the calibration graph was 10–1500 ng mL −1, with a detection limit of 5 ng mL −1. The precision of the method, expressed as relative standard deviation, ranged between 2.1% and 4.5%. After a sample treatment consisting on a conventional extraction, the method was applied to the determination of DNA in several samples from different biological materials.

Stopped-flow and kinetic-fluorimetric determination of quinalphos in water samples

The hydrolysis of the pesticide quinalphos in basic medium was kinetically followed and the measurement of the reaction rates allowed us to develop two kinetic-fluorimetric methods. In one of them the mixing of the reagents was directly performed in the measurement cell and, in the another one, the stopped-flow mixing technique was used. The reaction was completed in 100 s after the reactants were mixed and it allowed the simple application of the proposed methods to routine analyses of the pesticide. The sensitivity of the methods was very high, being the detection limits 50 and 140 ng mL −1 for the manual procedure and the stopped-flow mixing technique, respectively. Both methods were compared using regression with uncertainties in both axes. The effect of the presence of several pesticides in the determination was tested. A solid-phase extraction process was also developed for the application of the methods to diverse waters samples. The proposed kinetic-fluorimetric methods were applied to the determination of quinalphos in drinking water, well water and river water, with very satisfactory results.

Determination of soluble phosphates in water samples using ytterbium(III) and dynamic measurements of light scattering intensity at long wavelength

A selective and fast method has been developed for the determination of phosphates by measuring the formation of ytterbium(III) phosphate through the variation of the light scattering intensity with time. The low solubility of this compound causes an efficient dispersion of the radiation at 490 nm, which is measured at 980 nm using the second-order grating effect. This approach minimizes potential background signals from the sample matrix. The initial rate of the system is automatically obtained in only 0.5 s by stopped-flow mixing technique. The variable optimization study has been carried out using univariate and multivariate methods. The dynamic range of the calibration graph is 0.09–7.9 mmol L −1 (Pearson's correlation coefficient = 0.9999) and the detection limit is 0.03 mmol L −1. The precision of the method, expressed as relative standard deviation, is 2.3%. The study of the potential interference of different inorganic anions showed that arsenate is the main interferent, although it is tolerated in a molar ratio of 5.5. The method has been satisfactorily applied to the determination of soluble phosphates in tap, ground and river water using a previous preconcentration step with a Dowex 1 (1 × 4–400) anionic resin. A 500-fold concentration has been achieved, which has allowed to decrease the detection limit up to 60 mmol L −1. The recovery range is 97.5–102.5%. The results obtained are consistent with those obtained with the standard molybdenum blue method.

Simplex optimization of the variables affecting the micelle-stabilized room temperature phosphorescence of 6-methoxy-2-naphthylacetic acid and its kinetic determination in human urine

This article reports the kinetic determination of 6-methoxy-2-naphthylacetic acid (6-MNA), the major metabolite of nabumetone, from micelle-stabilized room temperature phosphorescence (MS–RTP) measurements made by using the stopped-flow mixing technique. This methodology allows one to determine analytes in complex matrices without the need for a tedious separation process. It also shortens analysis times substantially. The proposed method uses simplex methodology to optimize the chemical and instrumental variables affecting the phosphorescence. It was applied to the determination of 6-MNA in human urine. The maximum phosphorescence signal is obtained within only 10 s after the sample is prepared. The maximum slope of the kinetic curve, which corresponds to the maximum rate of the phosphorescence development, is measured at λ ex = 273 nm and λ em = 516 nm. Least-squares regression was used to fit experimental data, and the detection limit, repeatability, and standard deviation for replicate samples were determined.

Usefulness of Ytterbium(III) as Analytical Reagent for Total Sulfite Determination in White Wine Samples

Ytterbium(III) is used as reagent for the determination of sulfite by measuring the formation of the Yb(III)-sulfite complex through the variation of the light scattering intensity with time. The low solubility of this complex causes an efficient dispersion of the radiation at 490 nm, which is measured at 980 nm. Each kinetic datum is automatically obtained in only 0.5 s by stopped-flow mixing technique. The application of the initial rate method using a long emission wavelength minimizes the potential interference of fluorescent background signals from the sample matrix. The dynamic range of the calibration graph is 1-250 microg/mL, and the calculated detection limit is 0.35 microg/mL. The precision, expressed as relative standard deviation, is <6%. The method has been applied to the determination of total sulfites in white wine samples, which requires only the sample dilution and the use of two aliquots to improve selectivity. However, the matrix effect found for red wines precludes the application of the method to the direct analysis of these samples. Analytical recoveries ranged from 96.0 to 106.7%. The results obtained with the proposed method agreed with those provided by the p-rosaniline method. Unlike this method, in which toxic reagents are required, the use of ytterbium(III) as analytical reagent shows the advantage of its low acute toxic rating.

Simplex optimization and kinetic determination of nabumetone in pharmaceutical preparations by micellar—stabilized room temperature phosphorescence

The present method described the kinetic determination of nabumetone, a non-steroidal anti-inflammatory drug, by means of micellar-stabilized room temperature phosphorescence (MS-RTP), using the stopped-flow mixing technique. This methodology enables us to determine analytes in complex matrices without the need for a tedious separation process, as well as greatly diminishes the time for the analysis. Firstly, chemical and instrumental variables affecting the rate of phosphorescent development and the intensity of the signal, were found using a simplex optimization procedure. As application, nabumetone was determined in commercial Spanish pharmaceutical preparations. With the proposed method, the maximum signal of phosphorescence appears in only 10 s once the sample has been prepared, and the maximum slope of the kinetic curve, corresponding with the maximum rate of the phosphorescence development, was measured at λ ex = 271 nm and λ em = 520 nm. The overall least-squares regression to find the straight line that fitted the experimental data, the detection limit, the repeatability and the standard deviation for replicate sample, were also determined. The proposed method was validated versus a HPLC method with satisfactoty results.

Homogeneous stopped-flow fluorimmunoassay for gliadin determination in food samples

A homogeneous competitive immunoassay for gliadin determination is described for the first time. Gliadins are labeled with a terbium(III) chelate to form the tracer which competes with unlabeled gliadins for the active sites of anti-gliadin antibodies. The terbium(III) is released from the tracer by means of an “acidic enhancement solution” and the initial rate of the dissociation reaction is monitored using the stopped-flow mixing technique. Initial rate values obtained are different depending upon whether tracer is free or bound to antibody which can be correlated with the analyte concentration. The use of the fixed-time method has been also assayed, but it is not suitable as the results depend on the sample matrix. The detection limit of the assay using standards is 4 ng ml −1, which is equivalent to 4 μg g −1 in food samples, and the dynamic range of the method is 0.008–0.36 μg ml −1. The precision, expressed as the percentage of relative standard deviation, was 5–6.2%. The method has been applied to the analysis of gluten-containing and gluten-free food samples, with recoveries ranging from 83.8 to 115.1%.

Selective kinetic determination of amikacin in serum using long-wavelength fluorimetry

A simple and rapid method for the determination of the antibiotic amikacin, involving the use of a long-wavelength fluorophor, namely indocyanine green, (ICG) is presented. The dye is oxidised by cerium(IV) in acidic medium, resulting in a sharp decrease of the fluorescence, but this fluorescence quenching is inhibited in the presence of amikacin, which can be ascribed to the formation of an ion pair between the fluorophor and the analyte. The initial rate of the system is monitored at λex: 765nm and λem: 812nm as excitation and emission wavelengths, respectively, using the stopped-flow mixing technique, which makes the method applicable to automatic routine analysis. Each measurement is obtained in only 2–3s. The method presents a detection limit of 0.02μgm1−1 in standard solutions, which corresponds to 2.5μgml−1 in serum samples. The precision is in the range 4.8–6%. The good selectivity of the method allows amikacin to be determined in the presence of other antibiotics, including other aminoglycoside antibiotics, in serum. The recoveries obtained from the analysis of different samples were in the range 89.4–104.7%.

Fast kinetic determination of 1-naphthylacetic acid in commercial formulations, soils, and fruit samples using stopped-flow phosphorimetry.

A kinetic method has been developed for the determination of 1-naphthylacetic acid by means of micellar-stabilized room temperature phosphorescence (MSRTP) using the stopped-flow mixing technique. The main feature of this system is that it diminishes the time required for the deoxygenation of the micellar medium and for the phosphorescence development. Phosphorescence enhancers such thallium(I) nitrate, sodium dodecyl sulfate (SDS), and sodium sulfite were optimized to obtain maximum sensitivity. The pH was also optimized as it strongly affects the luminescent properties of 1-naphthylacetic acid. A pH of 6.6 was selected as adequate for the phosphorescence development. The kinetic curve of 1-naphthylacetic acid phosphorescence was scanned at lambda(ex) = 278 nm and lambda(em) = 490 nm, and the maximum rate of phosphorescence was taken as the analytical signal. This was obtained by calculating the maximum slope of the curve in an interval of 3.6 s as it provided a good noise-to-signal ratio. This method permitted the determination of 1-naphthylacetic acid throughout a concentration range of 100-1800 ng mL(-1) with high precision (relative standard error = 0.91% and relative standard deviation = 2.30%; 1-naphthylacetic acid concentration = 800 ng mL(-1)). According to the Clayton criterion, the detection limit was 45 ng mL(-1). The same limit resulted in 39.3 ng mL(-1) when the error propagation theory was applied. The applicability of the method was successfully demonstrated by determining 1-naphthylacetic acid in different kind of samples, such as phytosanitary products, soils, pears, and apples. Recovery values not significantly different from the nominal content or the spiked amount were found for these determinations.

Synthesis, characterization, and molecular structures of nitrosyl nitrito complexes of osmium porphyrins: Disproportionation of nitric oxide in its reaction with Os(P)(CO) (P = porphyrinato dianion)

The Os(P)(NO)(ONO) compounds (P = TTP, TMP, OEP, TmTP; TTP = 5,10,15,20-tetra-p-tolylporphyrinato dianion, TMP = 5,10,15,20-tetramesitylporphyrinato dianion, OEP = octaethylporphyrinato dianion, TmTP = tetra(m-tolyl)porphyrinato dianion) have been prepared from the reaction of the precursor carbonyl complexes Os(P)(CO) with excess nitric oxide. Nitrous oxide was detected as a by-product of the reaction. The IR spectra of the Os(P)(NO)(ONO) compounds (as KBr pellets) reveal bands in the 1790–1804 cm–1 range that are assigned to υNO. The IR spectra also reveal two new bands for each complex in the 1495–1531 and 913–962 cm–1 ranges indicative of O-bound nitrito ligands. The linearity of the bound NO groups and the O-binding of the trans nitrito ligands in the Os(P)(NO)(ONO) complexes are evident in the single-crystal X-ray crystal structures of the TTP and TMP derivatives. The kinetics of the reaction were studied by stopped-flow mixing techniques. Spectroscopic analysis of rapidly mixed solutions of Os(P)CO and NO in toluene showed a biphasic approach to the Os(P)(NO)(ONO) and N2O products, owing to the starting material Os(P)CO scavenging CO formed during the reaction to give Os(P)(CO)2 (KCO = 106 M–1). The dicarbonyl was the only transient species observed. It is proposed that the rate-determining step of the reaction leading to Os(P)(NO)(ONO) is NO displacement of CO from Os(P)(CO) via initial formation of an unstable 19 electron Os(P)(NO)(CO) intermediate.Key words: osmium, nitric oxide, X-ray, nitrosyl, porphyrin, kinetics.

Chemiluminescence determination of citrate and pyruvate and their mixtures by the stopped-flow mixing technique

A novel kinetic chemiluminescent method using the stopped-flow mixing technique has been investigated for the rapid and sensitive determination of citrate and pyruvate. The method is based on a tris(2,2′-bipyridiyl)ruthenium(III) (Ru(bpy) 3 3+) chemiluminescence (CL) reaction. Ru(bpy) 3 3+ was generated in the mixing chamber by oxidising tris(2,2′-bipyridyl)ruthenium(II) with cerium(IV). After selecting the best operating parameters, calibration graphs were obtained over the concentration ranges 0.38–38 μg ml −1 and 8.7–1300 ng ml −1 for citrate and pyruvate, respectively. The limits of detection were 0.1 μg ml −1 for citrate and 0.3 ng ml −1 for pyruvate. Based on the differential rate of the chemiluminescent reaction corresponding to citrate and pyruvate, a very simple kinetic procedure was developed for the simultaneous determination of both compounds. Mixtures of citrate and pyruvate in ratios between 15:1 and 1.5:1 were satisfactorily resolved. The proposed method was successfully applied to the determination of citrate in pharmaceutical formulations, pyruvate in animal blood serum and both compounds in human urine.

Stopped-flow Fluorescence Polarization Immunoassay

A general survey of the analytical application of kinetic methodology in fluorescence polarization immunoassay (FPIA) is presented. Stopped-flow mixing technique (SF) allows the initial rate of the immunochemical reaction between the tracer and the antibody to be obtained, which is used as the analytical parameter instead of the equilibrium signal used in conventional FPIA. The instrumentation required is described and the features of the analytical methods proposed are compared with those obtained by conventional FPIA. The usefulness of SF-FPIA for routine screening in clinical, environmental and food analysis is discussed.