Single Sample Solution Research Articles

Droplet size affects the degree of separation between fluorescence-positive and fluorescence-negative droplet populations in droplet digital PCR.

In droplet digital polymerase chain reaction (ddPCR) tests, a single sample solution is divided into many water-in-oil droplets. At the endpoint of PCR amplification, individual droplets are classified as either fluorescence-positive (FL(+)) or fluorescence-negative (FL(-)) droplets based upon their fluorescence amplitudes. Populations of FL(+) and FL(-) droplets can be seen in the histogram of fluorescence amplitude. The absolute copy number of a target molecule can be calculated from the fraction of FL(+) droplets relative to the total droplet number analyzed using Poisson statistics. It is crucial that the population of FL(+) droplets can be distinctly separated from that of the FL(-) droplets for accurately estimating the FL(+) droplet fraction and the absolute copy number. However, the distinct separation of the two populations is often impaired in actual ddPCR tests. Although many factors have been suggested to affect population separation, no study has addressed whether the droplet size influences the degree of separation. In this study, we compared the degrees of separation for ddPCR runs with three different droplet sizes. The experimental results showed an increasing degree of separation with decreasing droplet size. This discovery will potentially guide researchers to use smaller droplets in ddPCR to achieve higher accuracy and precision.

Multiplexed analysis of molecular and elemental ions using nanowire transistor sensors

An integrated sensor chip with silicon nanowire ion-sensitive field-effect transistors for simultaneous and selective detection of both molecular and elemental ions in a single sample solution is demonstrated. The sensing selectivity is realized by functionalizing the sensor surface with tailor-made mixed-matrix membranes (MMM) incorporated with specific ionophores for the target ions. A biomimetic container molecule, named metal-organic supercontainer (MOSC), is selected as the ionophore for detection of methylene blue (MB+), a molecular ion, while a commercially available Na-ionophore is used for Na+, an elemental ion. The sensors show a near-Nernstian response with 56.4 ± 1.8 mV/dec down to a concentration limit of ∼1 μM for MB+ and 57.9 ± 0.7 mV/dec down to ∼60 μM for Na+, both with excellent reproducibility. Extensive control experiments on the MB+ sensor lead to identification of the critical role of the MOSC molecules in achieving a stable and reproducible potentiometric response. Moreover, the MB+-specific sensor shows remarkable selectivity against common interfering elemental ions in physiological samples, e.g., H+, Na+, and K+. Although the Na+-specific sensor is currently characterized by insufficient immunity to the interference by MB+, the root cause is identified and remedies generally applicable for hydrophobic molecular ions are discussed. River water experiments are also conducted to prove the efficacy of our sensors.

Molecularly imprinted polymers for the selective detection of multi-analyte neurotransmitters

This paper reports on the development of electrochemical sensors for the detection of multiple neurotransmitters using molecularly imprinted polymers (MIPs). Pyrrole (PPy) and o-phenylenediamine (o-PD) were used as functional monomers for the MIP sensor development, and the characteristics of those sensors were analyzed. Specifically, we demonstrate a selective detection of dopamine (DA), norepinephrine (NE), and epinephrine (EP) by applying differential pulse voltammetry (DPV) to each uniquely developed MIP-based sensor. Furthermore, the selectivity of the analyte was quantified based on the sensitivity matrix. Our results demonstrate that all MIP sensors possessed higher sensitivity than non-imprinted (NIP) sensors due to the unique molecular receptors. The detection limits of the developed MIP sensors were less than 1.3×10−5M. The uniqueness of the cross-reactivity in the DA-imprinted and EP-imprinted sensors demonstrate the possibility of implementing a multi-analyte sensing platform that can detect multiple neurotransmitters simultaneously from a single sample solution.

One-Step Ligand Immobilization and Single Sample Injection for Regeneration-Free Surface Plasmon Resonance Measurements of Biomolecular Interactions.

Surface plasmon resonance (SPR) has been well established as a method of choice for label-free kinetic measurements of biomolecular interactions. The conventional approach involves multiple injections of an analyte of different concentrations into a fluidic channel covered with a fixed ligand density. Optimization of the experimental conditions and assessment of the data quality can be complicated by issues such as disruption of the ligand structure by the regeneration step and the limited availability of the sample solution. By sequentially closing fluidic channels on a five-channel SPR instrument, different densities of a ligand can be immobilized and determined in one step. With a subsequent injection of a single sample solution, SPR sensorgrams can be simultaneously collected to yield binding and dissociation rate constants (ka and kd) and dissociation constant (KD) between the ligand and analyte. For biomolecular interactions that obey the Langmuir isotherm, we show that the fidelity of the kinetic data can only be reliably confirmed when there exists a strong linear correlation between the SPR signals and the ligand densities. The use of a multichannel SPR instrument also obviates the regeneration step, allowing the binding kinetics between the green fluorescent protein and its antibody to be measured. In comparison to the conventional approach, the method simplifies the experimental procedure, reduces costs associated with sensor chips and biological samples, expedites kinetic measurements, and allows affinity constants to be determined more straightforwardly.

Perspectives on Method Validation: Validation is a Multistep Process with USP Regulatory Guidelines at each Step

<b>Perspectives on Method Validation</b>: Validation is a Multistep Process with USP Regulatory Guidelines at each Step

Rapid microfluidic separation of magnetic beads through dielectrophoresis and magnetophoresis

We present the design and fabrication of a new microfluidic device in which the dielectrophoresis and magnetophoresis phenomena were used for the separation of the superparamagnetic microbeads of different sizes. By exploiting the fact that two different particles can exhibit different dielectrophoretic force-frequency spectra, we utilize this device to perform multiplex detection from a single sample solution. We found the transition frequency range for 1, 2.8, and 4.5 microm magnetic beads using our device. Bead-based analysis revealed that a high separation efficiency ( approximately 90%) could be obtained from a single sample solution containing both 4.5 and 2.8 microm beads. The average flow velocity of the beads was maintained at 9.8 mm/s, enabling fast analysis with a smaller amount of reagents. The magnetic field distribution on the beads and the bead flow at the channel cross section for different dielectrophoretic conditions was obtained using CFD-ACE(+) simulation. Issues relating to the fabrication and operation of the device are discussed in detail. Finally, we demonstrated the feasibility of parallel detection/trapping of different beads on the same chip. This separation approach offers the performance of multiplex analysis in lab-on-a-chip devices.

Nanostructured Polyelectrolyte-based System as a Toolbox for Metal Ions Detection

The capability of certain heavy metal ions to induce fluorescence decrease by a quenching mechanism suggested us to design and build a sensor potentially tunable for different ions at different concentrations. We propose a quenching-based sensor exploiting a nanostructured architecture in which fluorescent molecules (the sensing probe) are entrapped to recognize a specific analyte (heavy metal ions) through an optical transduction. The polyelectrolyte nanostructured system, named nanocapsule, improves the fluorophore-ion quenching sensitivity allowing a micromolar detection. Furthermore we couple our sensor with an electrical device in order to refine the sensing procedure: the electric field created allows a metal ions spatial gradient, necessary to detect a specific element on a single sample solution, avoiding a comparative analysis with an intensity reference value. Results obtained will show the advantages and the potentialities of our system as a smart toolbox for metal ions detection.

Formation constant calculation for non-labile complexes based on a labile part of the metal-ligand system. A differential pulse polarographic study at fixed ligand to metal ratio and varied pH: application to polarographically inactive complexes

A new method of speciation by differential pulse polarography (DPP) is described. It is focused on non-labile complexes on the DPP time scale. By solving mass-balance equations - which are written for the labile part of a metal-ligand system - free metal, free ligand and non-labile complex concentrations are calculated. These are used for calculation of formation constants of non-labile complexes for each datum point. Each datum point is obtained during titration of a single sample solution by an addition of a standard sodium hydroxide solution. Where possible, the determinations were carried out in such a way that results from polarography and glass electrode potentiometry have been simultaneously obtained from a single experiment. The theoretical and analytical procedures as well as computer program (written to calculate formation constants directly from the polarographic data) were verified by experiment. Three metal ions, viz. Pb II, Cd II, and Bi III, were studied with the ligand N, N′,N″-tris(hydroxyethyl)triazacyclononane and formation constants, expressed as log K 1, were found to be 12.22, 10.61 and 16.36 from polarography, respectively. Results obtained for lead and cadmium are compared with those obtained from potentiometry. The validity of the proposed method and the application of DPP for polarographically inactive complexes are demonstrated.

A systematic and practical method of liquid chromatography for the determination of Sr and Nd isotopic ratios and REE concentrations in geological samples

A systematic and practical method for the determination of the concentration of the isotopic composition of Sr and Nd and rare-earth elements (REE) from a single sample solution has been developed by combining and improving classical cation-exchange chromatography (CCEC) and modern high-performance liquid chromatography (HPLC). The first step was used to separate Sr and the REE group from a dissolved rock solution by CCEC with a column of large volume and a combination of HClHNO 3 as an eluent. The second step was used to separate individual REE components present within the REE group by HPLC, simultaneously quantify them with a post-column reaction detector, and collect Nd with a programmable fraction collector. Separated Sr and Nd fractions were then purified by another CCEC with a column of small volume and HCl as an eluent prior to the mass spectrometric analysis. The analytical results obtained for a series of standard rocks showed a good agreement with recommended values to within ± 10% for REE quantification at the 95% confidence level and ±0.004% (1σ) for the 87 Sr 86 Sr and 143 Nd 144 Nd isotopic ratios.

Comparison of the TRAACS 800 with AOAC Methods for Calcium and Phosphorus in Feeds

Abstract Existing automated methods for calcium and phosphorus In animal feeds have been upgraded to permit determination of protein, calcium, and phosphorus simultaneously from a single sample solution running on a new generation of automated instruments. Minor changes and improvements in chemistry are described, and results using the new method are compared to results using AOAC official atomic absorption (calcium) and gravimetric (phosphorus) methods. For calcium, Mests showed no significant difference at the P ≤ 0.01 level over a range of concentration levels from 0-25%. For phosphorus, Mests showed no significant difference at the P ≤ 0.01 level over concentration ranges of 0 - 2% and 2 - 6%. Significant differences did exist at a concentration range of 6-18%. The small existing bias In these ranges was shown to be due to irregularities In the phosphorus standard curve fit, caused by protein and/or calcium components in the combined standard solutions.

Extraction of geological materials with mineral acids for the determination of arsenic, antimony, bismuth and selenium by hydride generation atomic absorption spectrometry

Data are presented for the hydride generation atomic absorption spectrometric determination of arsenic, antimony, bismuth and selenium in stream sediments and a certified reference material (CPB-1 Lead Concentrate) using six different extraction media, viz., hydrochloric acid; nitric acid; 7 M nitric acid; nitric acidsulphuric acid (9 + 1); aqua regia; and reversed aqua regia (HNO3+ HCl, 3 + 1). Extraction with 7 M nitric acid was performed in test-tubes placed in a drilled out heating block (110 °C, 3.5 h). All other extractions were carried out overnight in 50-ml calibrated flasks placed in a water-bath. Bismuth was extracted quantitatively by all the extraction media. Arsenic and selenium were extracted completely by each of the media used, except for hydrochloric acid. Antimony was extracted completely by only two of the media, viz., aqua regia and reversed aqua regia. Selenium is converted to the +4 oxidation state on extraction with aqua regia and reversed aqua regia, i.e., selenium(VI) is reduced to selenium(IV), and the lower oxidation states are oxidised to selenium(IV). Hence the extracts can be analysed for selenium without pre-reduction to selenium(IV), which is necessary for hydride generation atomic absorption spectrometry. The nitric acid-hydrochloric acid mixtures used bring arsenic, antimony, bismuth and selenium quantitatively into solution and are, therefore, suitable for the sequential or simultaneous determination of these elements in a single sample solution. The speciation of selenium in stream sediments and surface waters is discussed.

Selecting the normal population with the largest mean when the variances are bounded

A multiple decision approach to the problem of selecting the population with the largest mean was formulated by Bechhofer (1954), where a single-sample solution was presented for the case of normal populations with known variances. In this paper the problem of selecting the normal population with the largest mean is considered when the population variances are unequal and unknown but are constrained only to be less than a specified upper bound. It is demonstrated that a slight modification of Bechhofer' s procedure will suffice to ensure the probability requirements under this simple constraint for cases of practical interest.

Determination of silver, bismuth, cadmium, copper, iron, nickel and zinc in lead- and tin-base solders and white-metal bearing alloys by atomic-absorption spectrophotometry

A simple atomic-absorption spectrophotometry method is described for the determination of silver, bismuth, cadmium, copper, iron, nickel and zinc in lead- and tin-base solders and white-metal bearing alloys, with use of a single sample solution. The sample is dissolved in a mixture of hydrobromic acid and bromine, then fumed with sulphuric acid. The lead sulphate is dissolved in concentrated hydrochloric acid. The method is particularly suitable for the determination of silver and bismuth, which are co-precipitated with lead sulphate. The other elements can also be determined after removal of the lead sulphate by filtration.

Analytical characterization of CuInS 2 semiconductor material

Systematic analytical procedures have been developed for determination of the stoichiometry of CuInS 2 and estimation of trace elements, including dopants and impurities, in the material. Samples of CuInS 2 are digested in an oxidizing acid to ensure completely transformation into Cu 2+, In 3+ and SO 4 2− ions. The stoichiometry determination is made sequentially by controlled potential electro-deposition of copper, followed by its EDTA titration, titrimetric determination of indium and gravimetric determination of sulphate, in a single sample solution. The relative errors for the determination of Cu and In are found to be −0.08% and +0.11% respectively, fulfilling the requirement for accurate stoichiometry assessment; that for S is −0.66%, which though rather high is still acceptable. For the determination of trace elements in CuInS 2, multistage combined procedures are employed. Cu in the sample solution is removed by electro-deposition and In by extraction of HInBr 4 with isopropyl ether, then most of the trace elements are finally determined by atomic-absorption spectrometry, and the rest by neutron-activation analysis. All the steps involved in the procedures have been optimized by using radioisotopes as tracers. By the procedures developed, a wide range of trace elements in CuInS 2, down to submicrogram level, can be determined.

Determination of selenium, copper, lead and cadmium in biological materials by differential pulse stripping voltammetry

A simple and rapid stripping voltammetric method is described for the determination of selenium, copper, lead and cadmium in a single sample solution. The method utilizes differential pulse cathodic stripping voltammetry for the determination of selenium and differential pulse anodic stripping voltammetry for the other three elemetns. The use of a deposition potential of −0.3 V vs. SCE allows the determination of selenium in the presence of interfering ions at 1000-fold concentrations. Subsequent oxidation of the selenium to the electro-inactive selenium(Vi) permits direct determination of the other three elements in the same solution. Application of the method to NBS bovine liver and some fish samples proved to be satisfactory. The limits of detection obtained by this method were 0.1 μg l −1 for selenium, 0.5 μg l −1 for copper and 0.01 μg l −1 for lead and cadmium. Determination of the four elements takes about 90 min.

Rapid Screening Procedure for the Determination of Preservatives in Ground Beef: Sulfites, Benzoates, Sorbates, and Ascorbates

A procedure is described for the examination of large numbers of ground beef samples for the 4 most commonly used preservatives. A single sample solution is prepared and aliquots are taken for the various tests. Sulfite is determined colorimetrically with acid-bleached prosaniline. Ascorbic acid is titrated with 2,6-dichloroindophenol. Benzoic and sorbic acids are measured by ultraviolet absorption. Negative samples are rapidly sorted out in this procedure and positive samples are completed for quantitative determination of the preservative. Recoveries are 95% for sodium sulfite, 103% for sodium benzoate, 90% for potassium sorbate, and 81% expected recovery for sodium ascorbate. The limit of detection is 0.005% for potassium sorbate, and 0.001% for the 3 other perservatives.

A Bayesian Procedure for Selecting the Largest of k Normal Means

A Bayesian single sample solution to the problem of selecting the largest of k normal means is given. The solution appears robust with respect to variations in the prior distribution and parameters of the model.

A composite scheme for the analysis of steels by atomic-absorption spectroscopy using the air-acetylene flame

A composite scheme is described for the determination of chromium, molybdenum, manganese, nickel and copper in all types of steel. The scheme is based on a single sample solution containing 1 g of steel per 100 ml. Recent developments in technique have enabled all these elements to be determined with the air-acetylene flame and the scheme can be extended as required to include other elements such as lead and cobalt.

Determination of plutonium and uranium in mixed oxide fuels by sequential redox titration

The use of a sequential determination of uranium and plutonium in a single sample solution results in a saving in analysis time and apparatus requirements. The method starts with U(IV) and Pu(in) in a mixture of sulphuric and nitric adds. Titration with dichromate, using amperometry at a pair of polarizable electrodes, produces two well-defined end-points corresponding to the sequential oxidation of U(IV) to U(VI) and Pu(III) to Pu(IV). The quantitative oxidation of U(IV) to U(VI) is achieved via the action of Pu(IV) as intermediate, and is dependent upon establishing conditions which favour rapid reaction between U(IV) and Pu(IV). The method is precise and accurate. With Pu-U mixtures containing between 15 and 30% plutonium the precision (3σ) of the Pu: U ratio results is ±0.6% on samples containing 100–120 mg of plutonium plus uranium. Iron and vanadium interfere quantitatively with plutonium, copper interferes non-quantitatively with uranium, and gross amounts of molybdenum mask the uranium end-point.

Rapid method for analysis of Portland cement

A simple, rapid, complete and accurate method for analysing Portland cement, by using a single sample solution, is described.Silica is determined by a combined gravimetric-colorimetric method, while the remaining components are determined either spectrophotometrically (iron, titanium, phosphorus and manganese(III) oxides), complexometrically (sulphur trioxide, aluminium, calcium and magnesium oxides) or flame photometrically (sodium and potassium oxides). A feature of the method is the procedure for determining aluminium. The method involves the use of hexamine for adjusting pH, thereby preventing the hydrolysis of the titanium-EDTA complex from the corresponding EDTA-metal complexes; it also involves the use of fluoride ions for releasing the equivalent amount of EDTA combined with both aluminium and titanium. The results of analyses of six standard samples of Portland cement, by using this method, are compared with certified sample analyses of the U.S. National Bureau of Standards and confirm the accuracy of the method.With minor modifications the method can be applied to the analysis of cement raw materials such as limestones, clays and silicates.