Practical Detection Limit Research Articles

Method and apparatus using selected superparamagnetic labels for rapid quantification of immunochromatographic tests

A rapid method and instrumentation for quantification of immunochromatographic tests (ICT) are described. The principle and performance of the method was demonstrated by measuring the levels of human chorionic gonadotropin (hCG) present in urine. The test format was a sandwich assay using two distinct monoclonal antibodies directed against hCG. The first anti-hCG antibody was labeled with superparamagnetic particles whereas the second was immobilized as a narrow detection zone on a porous membrane. The human urine sample was mixed with superparamagnetic particles coated with the first anti-hCG antibody, and the mixture was allowed to migrate past the detection zone containing the second anti-hCG antibody. Capillary forces facilitated migration of the immune complexes along the porous membrane. The amount of superparamagnetic particle-labelled monoclonal anti-hCG bound to the detection zone was directly proportional to the amount of hCG present in the sample as detected by measuring magnetization in the detector coil. The method had a practical detection limit of 20 U/l (54 nM) of hCG per 5 μl of human urine and a linear range of three decades from 20 U/l to 10 000 U/l. In addition, the analysis was completed within less than 10 minutes. Thus, the test format should be suitable for fast detection and monitoring of a large variety of clinically important parameters and analytes.

Coupling of a vented column with splitless nanoRPLC-ESI-MS for the improved separation and detection of brain natriuretic peptide-32 and its proteolytic peptides

The circulating concentration of a biomarker for congestive heart failure, Brain (B-type) Natriuretic Peptide (BNP-32), is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of molecular specificity afforded by these assays has recently come into question as emerging studies indicate there are potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities. In order to better understand the molecular biology of BNP-32 as it relates to congestive heart failure, it would thus be advantageous to use a detection platform such as Fourier transform ion cyclotron resonance mass spectrometry. This high resolving power mass spectrometer can provide unparalleled molecular specificity and can facilitate identification and characterization of the various molecular forms across all disease states. Unfortunately, BNP circulates at low concentrations (as low as 3 fmol/mL). Thus, it will require a collaborative effort from a number of orthogonal front-end technologies to overcome the disconnect between the practical detection limits of this instrument platform and the physiological levels of BNP-32 and its alternative molecular forms. Herein, we begin optimization of these front-end techniques by first enhancing the conditions for online nanoLC-ESI-MS separations of BNP-32 and its proteolytic fragments. Through extensive analysis of various chromatographic parameters we determined that Michrom Magic C8 stationary phase used in conjunction with a continuous, vented column configuration provided advanced chromatographic performance for the nano-flow separations involving intact BNP-32 and its associated tryptic peptides. Furthermore, conditions for the tryptic digestion of BNP-32 were also studied. We demonstrate that the use of free cysteine as an alkylation quenching agent and a secondary digestion within the digestion scheme can provide targeted tryptic peptides with increased abundances. Combined, these data will serve to further augment the detection of BNP-32 by LC–MS.

Optical resonant sensors: a method to reduce the effect of thermal drift

The use of a whispering gallery mode (WGM) resonator with an ultrahigh quality factor Q is promising in highly sensitive, label-free, lab-on-a-chip sensor applications. We investigated a novel method of using the differential frequency of TE and TM modes to reduce the thermal noise baseline, which commonly limits the sensitivity of WGM sensors. We studied the temperature dependence of WGM based sensors and experimentally demonstrated the reduction of temperature fluctuation and thus a significant improvement in the practical sensor detection limit.

Determination of urinary bisphenol A by coacervative microextraction and liquid chromatography–fluorescence detection

Total (free plus conjugated) urinary BPA is currently being used to assess human exposure to this contaminant. This work proposes the use of coacervates made up of reverse micelles of decanoic acid for the microextraction of BPA, prior to its determination by liquid chromatography and fluorescence detection ( λ ex = 276 nm and λ em = 306 nm), with the aim of simplifying sample treatment and reducing analysis time and costs in epidemiologic studies. The procedure involves the enzymatic hydrolysis of 7 mL of urine and then the addition of decanoic acid (100 mg) and tetrahydrofurane (1 mL), conditions under which the coacervate (extractant phase, ∼167 μL) forms in situ and instantaneously. The overall procedure takes about 20 min and several samples can be simultaneously treated using conventional lab equipment (i.e. stirrers and centrifuges). Extractions were independent of the pH and temperature in the ranges studied (1–4 and 25–50 °C) rendering the method robust. Recoveries in samples ranged between 88 and 95% and the practical detection limit was 0.197 μg L −1, which is below the usual concentrations of BPA in urine (ranges reported 0.4–149 μg L −1). The actual concentration factor provided by the method was 38. The precision of the method, expressed as relative standard deviation, was 4.5%. The method was successfully applied to the determination of total BPA in urine from eight healthy volunteers. BPA was detected in all the samples at concentrations ranging between 4.03 and 49 μg L −1.

High Sensitivity Electrochemical Detection of Hydrogen Peroxide at a Cobalt Phthalocyanine-Modified Boron-Doped Diamond Electrode

A cobalt phthalocyanine-modified boron-doped diamond (CoPc-BDD) electrode was fabricated. The hydrogen-terminated BDD electrode surface was terminated with the pyridine moieties via photochemical modification with 4-vinylpyridine (4VP), followed by immobilization of CoPc on the surface by immersion of the 4VP-BDD samples in a CoPc solution. X-ray photoelectron spectroscopy studies suggested that coordination of the surface pyridine to the Co atom in CoPc contributes to the surface modification. Electrochemical detection of hydrogen peroxide at the modified electrode was also investigated. Cyclic voltammetry confirmed that the CoPc-BDD electrode exhibits catalytic activity for the electrochemical oxidation of H2O2. Using a flow-injection analysis-electrochemical detection (FIA-EC) system, CoPc-BDD electrode was estimated as being highly suitable for the detection of H2O2 with a practical detection limit in the order of several tens of nanomolars.

A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical Ultracentrifugation

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become an important tool for the characterization of the purity of protein therapeutics. The work presented here addresses a need for methods orthogonal to size-exclusion chromatography for ensuring the reliable quantitation of immunogenic oligomers, for example, in antibody preparations. Currently the most commonly used approach for SV-AUC analysis is the diffusion-deconvoluted sedimentation coefficient distribution c(s) method, previously developed by us as a general purpose technique and implemented in the software SEDFIT. In both practical and theoretical studies, different groups have reported a sensitivity of c(s) for trace oligomeric fractions well below the 1% level. In the present work we present a variant of c(s) designed for the purpose of trace detection, with customized Bayesian regularization. The original c(s) method relies on maximum entropy regularization providing the most parsimonious distribution consistent with the data. In the present paper, we use computer simulations of an antibody system as example to demonstrate that the standard maximum entropy regularization, due to its design, leads to a theoretical lower limit for the detection of oligomeric traces and a consistent underestimate of the trace populations by approximately 0.1% (dependent on the level of regularization). This can be overcome with a recently developed Bayesian extension of c(s) (Brown et al., Biomacromolecules, 8:2011-2024, 2007), utilizing the known regions of sedimentation coefficients for the monomer and oligomers of interest as prior expectation for the peak positions in the distribution. We show that this leads to more clearly identifiable and consistent peaks and lower theoretical limits of quantization by approximately an order of magnitude for some experimental conditions. Implications for the experimental design of SV-AUC and practical detection limits are discussed.

Adaptive Fiber-Optical Sensor System for Pico-Strain and Nano-Displacement Metrology

Adaptive fiber-optic interferometer which is based on multimode optical fiber as a sensor and diffusion holograms recorded in semiconductor photorefractive crystal CdTe:V without any electric field is developed. The interferometer sensitivity achieved is only 5.7 times less then highest sensitivity which is possible only in non-adaptive lossless classical interferometer. A practical detection limit is equal to Hz W nm 10 0 3 5 − × . , which allows to broadband detecting of an object’s displacement of order 0.2 nm or deformation of order 2 pε with using light sources having only 5 mW optical power.

Voltammetric behavior of multi-walled carbon nanotubes modified electrode-hexacyanoferrate(II) electrocatalyst system as a sensor for determination of captopril

Abstract The study of electrochemical behavior and determination of captopril, as an angiontensin-converting enzyme inhibitor, is reported on multi-walled carbon nanotube (MCNT) modified glassy carbon electrode (GCE) and hexacyanoferrate(II) (HCF) electrocatalyst. The cyclic voltammetric results indicate that MCNTs and HCF system can remarkably enhance electrocatalytic activity toward the oxidation of captopril in acidic solutions. It is leading to a considerable improvement of the anodic peak current for captopril, and allows the development of a highly sensitive voltammetric sensor for detection of captopril in pharmaceutical and clinical samples. The investigation of captopril oxidation on the iron oxide nanoparticles modified carbon paste electrode does not show any electrocatalytic effect on the oxidation of captopril, suggesting that iron oxide impurities in the MCNTs are not the active sites in captopril sensing. Under optimized conditions on MCNTs-HCF system, the proposed method with respect to other reported electrochemical methods shows wider dynamic range (0.5–600 μM) with suitable selectivity, practical detection limit of 0.2 μM and good precision (R.S.D.

Determination of Cd in urine by cloud point extraction–tungsten coil atomic absorption spectrometry

Cadmium concentrations in human urine are typically at or below the 1 μg L −1 level, so only a handful of techniques may be appropriate for this application. These include sophisticated methods such as graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry. While tungsten coil atomic absorption spectrometry is a simpler and less expensive technique, its practical detection limits often prohibit the detection of Cd in normal urine samples. In addition, the nature of the urine matrix often necessitates accurate background correction techniques, which would add expense and complexity to the tungsten coil instrument. This manuscript describes a cloud point extraction method that reduces matrix interference while preconcentrating Cd by a factor of 15. Ammonium pyrrolidinedithiocarbamate and Triton X-114 are used as complexing agent and surfactant, respectively, in the extraction procedure. Triton X-114 forms an extractant coacervate surfactant-rich phase that is denser than water, so the aqueous supernatant is easily removed leaving the metal-containing surfactant layer intact. A 25 μL aliquot of this preconcentrated sample is placed directly onto the tungsten coil for analysis. The cloud point extraction procedure allows for simple background correction based either on the measurement of absorption at a nearby wavelength, or measurement of absorption at a time in the atomization step immediately prior to the onset of the Cd signal. Seven human urine samples are analyzed by this technique and the results are compared to those found by the inductively coupled plasma mass spectrometry analysis of the same samples performed at a different institution. The limit of detection for Cd in urine is 5 ng L −1 for cloud point extraction tungsten coil atomic absorption spectrometry. The accuracy of the method is determined with a standard reference material ( toxic metals in freeze-dried urine) and the determined values agree with the reported levels at the 95% confidence level.

Sequential Injection Lab‐on‐Valve Procedure for the Determination of Amantadine Using Potentiometric Methods

AbstractAmantadine potentiometric detectors were developed, evaluated and incorporated in a SIA‐LOV manifold in order to accomplish the control of pharmaceutical formulations and urine. The electrodes incorporate α‐cyclodextrin as ionophore, dibutyl phthalate or 2‐fluorophenyl 2‐nitrophenyl ether as plasticizers and potassium tetrakis[3,5‐bis‐(trifluoromethyl)phenyl]borate (KTFPB) as cationic additive. The slope increased from 61.2 to 63.8 mV decade−1 and the practical limit of detection from 2.6×10−6 mol L−1 to 2.5×10−5 mol L−1 when the plasticizer was changed from 2‐fluorophenyl 2‐nitrophenyl ether to dibutyl phthalate. When incorporated in the flow‐manifold the membranes composed by dibutyl phthalate or with 2‐fluorophenyl 2‐nitrophenyl ether presented slopes and a practical limit of detection of 69.8 mV decade−1 and 1.5×10−4 mol L−1 or 73.7 mV decade−1 and 5.4×10−5 mol L−1, respectively. The electrode presented stable responses for over a year, and were highly selective concerning the representative species of the two sample matrices assayed as interferents. Comparison of obtained results with those provided by reference methods and recovery assays, revealed adequate accuracy for control assays.

High-performance liquid chromatography method for quantifying sphingomyelin in rat brain

A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile–methanol–water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl- sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025–0.4 mg/ml sphingomyelin ( R 2 > 0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.

Optimally electrochemically roughened silver substrates prepared in gold complexes-containing solutions for trace molecules detection

We report here the first optimally electrochemical method to prepare surface-enhanced Raman scattering (SERS)-active roughened silver substrates with bimetallic silver and gold nanoparticles. First, a gold substrate was roughened by a triangular-wave oxidation–reduction cycle (ORC) in an aqueous solution containing 0.1 mol/L KCl. Gold-containing complexes were found in the solution after the ORC treatment. Then a silver substrate was subsequently roughened by the similar ORC treatment using different parameters in the same solution. The SERS of R6G adsorbed on the optimal procedure-prepared substrate exhibits a higher intensity by one order of magnitude, as compared with that of R6G adsorbed on a normal method-prepared substrate. Moreover, the practical limit of detection (LOD) of R6G can be reduced by 5 orders of magnitude from 2 × 10 −10 to 2 × 10 −15 M. Further investigations indicate that surface morphologies of roughened Ag substrates and the oxidization states of Ag and the contents of Cl on the roughened Ag substrates are responsible for these improved SERS effects.

Optimization of a species-specific polymerase chain reaction assay for identification of Pentatrichomonas hominis in canine fecal specimens

To determine the optimum reaction conditions and detection limits of PCR assay for identification of Pentatrichomonas hominis in DNA extracted from canine feces. DNA extracted from feces of 4 dogs with diarrhea from which trichomonads were observed, 81 dogs that had feces submitted to a diagnostic laboratory, and 19 dogs residing in a laboratory animal facility. Optimum reaction conditions and absolute and practical detection limits of 2 P hominis 18S species-specific primer pairs were determined by use of an in vitro cultivated canine isolate of P hominis in the presence and absence of canine feces. The optimized PCR assay was applied to amplification of P hominis 18S rRNA genes from DNA extracted from the feces of dogs. Under optimized conditions, a primer pair was identified as able to detect as few as 1 P hominis organism/180-mg fecal sample. The PCR assay identified P hominis in diarrheic feces of 4 dogs in which trichomonads were seen by light microscopy. The P hominis genes were not amplified from other fecal samples examined. Molecular identification of P hominis in feces of 4 dogs with trichomonosis and diarrhea reported here validates the identity of this species in such infections. Sensitive and specific PCR amplification of P hominis 18S rRNA genes from DNA extracted from feces will directly facilitate studies examining pathogenicity of this trichomonad and enable differentiation of P hominis from other known or novel species of trichomonads that may infect the gastrointestinal tract of dogs.

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.

A novel real-time polymerase chain reaction method for the detection of pecan nuts in food

A real-time PCR-based method for the detection of the pecan (Carya illinoiensis) component in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pecan-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the putative gene for allergenic vicilin-like seed storage protein of pecan. The method was positive for 10 pecan varieties and negative for all other tested plant materials used in food industry, including walnut. The intrinsic detection limit of the method was 1 pg pecan DNA which corresponds to 1.2 haploid genome copies. Using a series of model pastry samples with defined pecan contents, a practical detection limit of 0.01% (w/w) pecan was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples; no discrepancies between the declared and detected pecan contents were found. The presented PCR method is useful for sensitive and selective detection of pecans in food samples and can be performed in one working day.

Novel surface-enhanced Raman scattering-active silver substrates containing visible light-responsible Tio2 nanoparticles

We report here the first electrochemically prepared surface-enhanced Raman scattering (SERS)-active silver substrates containing visible light-responsible TiO2 nanoparticles. The SERS-active Ag substrates with TiO2 nanoparticles were prepared by roughening the substrates using electrochemical oxidation–reduction cycles (ORC) in 0.1 M HCl aqueous solutions containing 1 mM visible light-responsible TiO2 nanoparticles. Based on these prepared substrates, the SERS of Rhodamine 6G (R6G) exhibits a higher intensity by one order of magnitude, as compared with that of R6G adsorbed on a roughened Ag substrate without TiO2 nanoparticles. Moreover, the practical limit of detection (LOD) of R6G can be reduced by five orders of magnitude from 2 × 10−10 to 2 × 10−15 M owning to the additional contribution of TiO2 nanoparticles to the whole SERS effects.

Determination of Silicate in Anthropogenically Impacted Natural Waters Using Flow Injection Spectrophotometry

An optimised flow injection (FI) method suitable for the determination of silicate in the presence of high concentrations of arsenate (216 µg As L -1 ) in anthropogenically impacted natural waters is presented. The proposed method has a practical limit of detection of 10 µg Si L -1 and typical RSDs of ≈1.5 % (n = 3) and a sample throughput of 40 samples h -1 . Strategies are presented for the removal of matrix interferences. The manifold incorporates a thiosulfate stream to remove arsenate (AsO4 3) by reduction to arsenite (AsO3 3) and two micro-columns containing a chelating resin (iminodiacetate) to remove metal ions and an anion exchange resin (Dowex 1X8, 200–400 mesh, Cl - form) to remove phosphate. The chelating column successfully removed matrix cations; Cu(II) at 0.1 mg L -1 , Ni(II), Co(II), Fe(II) and Fe(III) at 1.0 mg L -1 and Mn(II), Zn(II) and Pb(II) at 10 mg L -1 . The anion exchange column effectively removed phosphate interference by complexing up to 1.5 mg P L -1 and had no effect on the silicate response. A linear calibration was obtained with the optimised manifold in the range 10 - 1000 µg Si L -1 (R 2 = 0.998). This method was applied to the determination of Si in the Tamar Estuary, an area of historical mining activity, and the results compared well with those from a segmented flow analyser standard method with spectrophotometric detection (P = 0.05; tcal = 1.44 and tcrit = 2.14).

Determination of drinking water-arsenic in nano gram level using flow injection analysis system atomic absorption spectrometry and an interpretation for the cause behind contamination

The ground water in arsenic infested zone, West Bengal, India is characterised by high iron, arsenic, calcium, magnesium and bi-carbonate with low chloride and sulphate. The pH is generally greater than 7. In this pH medium, iron tends to precipitate as hydroxide and arsenic may also co-precipitate with ferric hydroxide. Addition of hydrochloric acid is necessary to dissolve all iron, prior to arsenic analysis by flow injection analysis system - atomic absorption spectrometric method. Addition of sufficient KI in HCl medium is necessary to reduce all As 5+ to As 3+ state before analysis. The average practical detection limit of the FIAS-AAS method is 0.035 pg/L. The method itself is highly sensitive and sensitivity is 0.07 μg/L which is far better than many other methods such as flame atomic absorption, hydride generation atomic absorption spectrometric method and from the conventional spectrophotometric method. Shallow tubewell or well shows the highest peak in the bar graph.

Study of Gas Exchange in Insects by Sensitive Laser Photoacoustic Spectroscopy

Although quantifying gas exchange in small insect species is of great biological interest, the progress in this field of research is hampered by the inability of most gas detectors to monitor the low emission rates from these insects. Recently, laser based photoacoustic spectroscopy (LPAS) has emerged as a highly sensitive technique that allows gases to be detected at sub‐ppb level. In this work, LPAS was used to study gas exchange in two different insect species. Firstly, loss of water by the minute Western Flower thrips (Frankliniella occidentalis, average weight 50 µg) was recorded by means of CO‐laser PAS. Water loss by a single thrips could be on‐line recorded with a practical detection limit of 30 ppb. Insects released H2O in rather regular patterns, possibly due to discontinuous respiration. These data represent on‐line H2O loss recordings of the smallest insects measured hitherto. In a second experiment, the inert tracer gas SF6 was used to determine the tracheal volume of Attacus atlas pupae. Insects were loaded with SF6 and subsequent release of this gas was monitored using CO2‐laser PAS. Based on the amount of released SF6, one has determined the tracheal volume of Attacus atlas pupae. The value obtained (170±53 µL/g) is in good correspondence with data obtained by other techniques. In addition, with this technique, respiration patterns can be recorded; the results show a good correspondence with the simultaneous recordings of CO2 release using a commercial CO2 analyser.

Semi-nested PCR detection of Babesia orientalis in its natural hosts Rhipicephalus haemaphysaloides and buffalo

Babesiosis has recently been recognized as an emerging infectious disease of buffalo in China. In order to investigate the epidemiology and enzootic potential of this parasite in Hubei province, we sought to develop a semi-nested PCR to detect Babesia orientalis in buffalo and the potential tick vector -Rhipicephalus haemaphysaloides by amplifying a specific 257 bp fragment of B. orientalis 18S rRNA gene. The practical limit of detection showed that it had high sensitivity and an approximate parasitemia of 0.00000012% was detected by the PCR system. The blood samples of 121 asymptomatic buffaloes collected from four babesia endemic counties and that of 71 asymptomatic buffaloes collected from three babesia free counties in Hubei province of China were examined for the presence of B. orientalis using both Wright–Giemsa stained blood smear and semi-nested PCR. Microscopic examination revealed that 5/121 animals were positive, whereas 24/121 animals were positive by the semi-nested PCR assay. Of 378 ticks ( R. haemaphysaloides) collected from buffaloes and examined by the semi-nested PCR, 35 were positive. The results showed that the semi-nested PCR was a useful method to investigate the epidemiology of buffalo babesiosis ( B. orientalis), which is widely distributed in Hubei province, China.