Photometric Detection System Research Articles

Abstract 11475: A Genetically Modified pH Resistant NCX1 Mouse Prevents Calcium Overload During Acidosis and is Cardioprotective

Acidosis inhibits the cardiac sodium(Na)/calcium(Ca) exchanger (NCX1), allegedly contributing to elevated cytoplasmic Na and Ca concentrations during ischemia. Upon reperfusion and restoration of normal pH, a reactivated NCX1 abruptly shifts into reverse (Ca influx) mode because of the abnormally high cellular Na concentration, thus producing massive Ca overload and cell damage. To test the dependence of ischemia/reperfusion (I/R) injury on NCX1’s pH sensitivity, we used CRISPR/Cas9 technology to create mice with a pH-resistant NCX1 by substituting Alanine for Histidine at position 165 (H165A). These mice live into adulthood with slightly reduced LV function (EF% H165A: 50 ± 5; Wildtype (WT): 67 ± 2; p<0.025, N=4-6). In enzymatically isolated adult ventricular myocytes, we used the patch clamp technique and voltage ramps from +80 to -120 mV to confirm that NCX1 current in H165A mice is resistant to pH reduction (from 7.4 to ~pH 6.4 by substituting 80 mM Na-Acetate for equimolar NaCl) when compared to WT animals (% reduction at +80mV: WT 77.7± 2.8, P<0.027 in 3 cells from 2 mice; H165A 0.21 ± 11.35, P=0.31 in 7 cells from 3 mice). We then performed I/R studies in explanted hearts perfused in Langendorff fashion with oxygenated Tyrode’s containing (in mM): 136 NaCl, 1.8 CaCl 2 , 5.4 KCl, 0.33 NaH 2 PO 4 , 1.0 MgCl 2 , 10 HEPES, and 10 glucose, pH 7.4, 37 o C. 20 minutes of stop-flow ischemia were followed by reperfusion for 120 minutes. Necrosis area (pixels 2 ) in WT was 26 ± 3 (N=11), compared to only 9 ± 1 (N=7) in H165A mice (P<0.0004). To compare effects of lower pH on isolated cardiomyocyte Ca, we used a photometric epifluorescence detection system to measure cytosolic Ca in enzymatically isolated ventricular myocytes loaded with the Ca indicator fura-2 AM (2 μM). When pH was lowered to ~6.4, Ca transients (ΔF340/F380) increased in WT from 0.6 to 0.9 (P < 0.0004, N=19 cells from 3 mice) but notably did not change in H165A (0.5 ± 0.03 @ pH 7.4, 0.5 ± 0.04 @ pH 6.4, P=0.307, N=21 cells from 3 mice). pH changes were verified in separate cells using the pH indicator SNARF-AM (10 μM). We conclude that eliminating pH sensitivity in NCX1 has major effects on intracellular Ca regulation, is cardioprotective, and a potential strategy for therapies in patients with prolonged ischemia prior to reperfusion.

One step multi-material 3D printing for the fabrication of a photometric detector flow cell

Optical detection is the most common detection mode for many analytical assays. Photometric detection systems and their integration with analytical systems usually require several assembly parts and manual alignment of the capillary/tubing which affects sensitivity and repeatability. 3D printing is an innovative technology for the fabrication of integrated complex detection systems. One step multi-material 3D printing has been explored to fabricate a photometric detector flow cell from optically transparent and opaque materials using a dual-head FDM 3D printer. Integration of the microchannel, the detection window and the slit in a single device eliminates the need for manual alignment of fluidic and optical components, and hence improves sensitivity and repeatability. 3D printing allowed for rapid design optimisation by varying the slit dimension and optical pathlength. The optimised design was evaluated by determining stray light, effective path length and the signal to noise ratio using orange G. The optimised flow cell with extended path length of 10 mm and 500 μm slit yielded 0.02% stray light, 89% effective path length and detection limit of 2 nM. The sensitivity was also improved by 80% in the process of optimisation, using a blue 470 nm LED as a light source.

Effect of Ethanol on the Adsorption of Volatile Sulfur Compounds on Solid Phase Micro-Extraction Fiber Coatings and the Implication for Analysis in Wine

Complications in the analysis of volatile sulfur compounds (VSC) in wine using solid-phase microextraction (SPME) arise from sample variability. Constituents of the wine matrix, including ethanol, affect the volatility and adsorption of sulfur volatiles on SPME fiber coatings (Carboxen- polydimethylsiloxane(PDMS); DVB-Carboxen-PDMS and DVB-PDMS), which can impact sensitivity and accuracy. Here, several common wine sulfur volatiles, including hydrogen sulfide (H2S), methanethiol (MeSH), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), diethyl disulfide (DEDS), methyl thioacetate (MeSOAc), and ethyl thioacetate (EtSOAc) are analyzed, using SPME followed by gas chromatography (GC), using a system equipped with a pulsed-flame photometric detection (PFPD) system, at various ethanol concentrations in a synthetic wine matrix. Ethyl methyl sulfide (EMS), diethyl sulfide (DES), methyl isopropyl sulfide (MIS), ethyl isopropyl sulfide (EIS), and diisopropyl disulfide (DIDS) are evaluated as internal standards. The absorption of volatile compounds on the SPME fiber is greatly affected by ethanol. All compounds exhibit a stark decrease in detectability with the addition of ethanol, especially between 0.0 and 0.5% v/v. However, the ratio of interested sulfur compounds to the internal standard becomes more stable when the total alcohol concentration exceeds 2%. EMS was found to best resemble DMS. EIS and DES were found to best resemble DMDS, MeSOAc, and EtSOAc. DIDS was found to best resemble DEDS, DMTS, H2S, and MeSH.

Lab on a Chip for the Colorimetric Determination of Nitrite in Processed Meat Products in the Jordanian Market.

Nitrite and Nitrate have been used extensively as additives in various meat products to enhance flavor, color, and to preserve the meat from the bacterial growth. High concentrations of nitrite can threat human health since several studies in the literature claim that nitrite is associated with cancer incidences, leukemia, and brain tumors. Therefore, it is vital to measure the nitrite concentrations in processed meat products. In this study, an in-lab miniaturized photometric detection system is fabricated to inspect the nitrite concentration in processed meat products in Jordan. The analytical performance of nitrite detection is evaluated based on three key statistical parameters; linearity, limit of detection, and limit of quantitation. Respectively, for the fabricated system, the three values are found to be equal to 0.995, 1.24 × 10−2 ppm, and 4.12 × 10−2 ppm. Adherence to Beer’s law is found over the investigated range from 2.63 ppm to 96.0 ppm. The developed system is utilized for photometric detection of nitrite in processed meat products available in the Jordanian market like pastrami, salami, and corned beef. In all of the analyzed samples, the nitrite content is found to be lower than 150 ppm, which represents the maximum allowable nitrite limit.

LOW COST ANALYZER FOR THE DETERMINATION OF PHOSPHORUS BASED ON OPEN-SOURCE HARDWARE AND PULSED FLOWS

The need for automated analyzers for industrial and environmental samples has triggered the research for new and cost-effective strategies of automation and control of analytical systems. The widespread availability of open-source hardware together with novel analytical methods based on pulsed flows have opened the possibility of implementing standalone automated analytical systems at low cost. Among the areas that can benefit from this approach are the analysis of industrial products and effluents and environmental analysis. In this work, a multi-pumping flow system is proposed for the determination of phosphorus in effluents and polluted water samples. The system employs photometric detection based on the formation of molybdovanadophosphoric acid, and the fluidic circuit is built using three solenoid micropumps. The detection is implemented with a low cost LED-photodiode photometric detection system and the whole system is controlled by an open-source Arduino Uno microcontroller board. The optimization of the timing to ensure the color development and the pumping cycle is discussed for the proposed implementation. Experimental results to evaluate the system behavior are presented verifying a linear relationship between the relative absorbance and the phosphorus concentrations for levels as high as 50 mg L-1.

Microfluidic chip-based analytical system for rapid screening of photocatalysts

A simple and efficient microfluidic chip-based analytical system for rapid screening of photocatalysts was developed. The catalyst screening system consisted of a microchip with multiple channels for parallel reactions, a UV light source, and a CCD camera-based photometric detection system for monitoring the photocatalytic reaction. A novel microfluidic introduction method for loading particle samples into chip microchannels was established using dry sample powders and wedge-structure channel design. With this method, multiple different photocatalyst samples could be quickly introduced into the microchip with good reproducibility without the need of additional pumps or valves. We applied the present system in the rapid screening of doping TiO2 photocatalysts in terms of their activity for methylene blue (MB) degradation under UV light irradiation. Ten parallel photocatalyst screening reactions were achieved within 15 min in the multi-channel chip. We also examined nine element doped TiO2 materials to investigate the doping effects of different elements on TiO2. Compared with conventional systems, the photocatalyst consumption (0.1mg) in the present system was significantly reduced at least 100 times. High reaction rate in chip microreactors was obtained with an increase of two orders of magnitude over bulk reactors. The miniaturization of the photocatalytic reaction on the microchip significantly improves the reaction rates, reduces the sample and reagent consumptions, and increases the throughput of screening for multiple catalyst samples in parallel. The present work provides a novel application for microfluidic chip-based analytical systems, as well as a rapid, highly-efficient and low-consumption method for screening of photocatalysts.

Application of direct-injection detector integrated with the multi-pumping flow system to photometric stop-flow determination of total iron

A novel direct-injection detector (DID) integrated with multi-pumping flow system (MPFS) for the photometric determination of iron is proposed. Paired emitter-detector diodes have been used as a photometric detection system. The sample and reagent were injected using appropriate solenoid pulse micro-pumps directly into the detection chamber where effective mixing occured. The use of proposed stop-flow detector considerably simplified the analytical procedure. The potassium thiocyanate has been chosen as a chromogenic reagent for photometric Fe(III) detection. The total volume of reagent and sample/standard solutions involved in the detection process was adjusted to the volume of the reaction-detection chamber.Calibration graph was found to be linear in the range up to 10mgL−1. The detection limit (3sb/S) was 0.15mgL−1. The repeatability (R.S.D.), calculated from 10 analyses of sample containing 5mgL−1 Fe(III), was 1.5% and the sample throughput 180 determinations per hour. The consumption of sample and reagent was 20μL each with the waste generation at the level of 0.24mL. The applicability of the proposed method to the determination of total iron in groundwater samples has been proved.The analytical parameters are compared to those obtained exploiting the MPFS system with typical configuration containing a confluence point and reaction coil.

Diagnosis of Helicobacter pylori bacterial infections using a voltammetric biosensor

The voltammetric assay of Helicobacter pylori DNA was investigated using a bismuth-immobilized carbon nanotube electrode (BCNE). The analytical cyclic voltammetry (CV) peak potential was obtained at a 0.4 V reduction scan, where the diagnostic optimum square-wave (SW) stripping working range was achieved at 0.72–7.92 μg/mL H. pylori DNA (11 points). A relative standard deviation of 1.68% (RSD, n = 5) was obtained with 3.2 mg/mL H. pylori DNA using a 240 s accumulation time. Under optimum conditions, detection limit was 0.06 μg/mL. The developed sensors can be used for clinical application in the 15th doubted human gastric tissues, since the patient's peak current increased a hundred times more than the negative healthy tissue did. The sensing time obtained was only two minutes, and the process was simpler compared to common PCR amplification and electrophoresis photometric detection systems.

Development of a Low-Cost SIA-Based Analyser for Water Samples

An automated multiparametric water analyser was developed and evaluated. The system was based on Sequential Injection Analysis and featured a photometric detection system comprising a tricolour RGB LED source and a photodiode. A program compiled in Visual Basic was used to control the SIA flow system, the LEDs, and the data acquisition and processing. The program loads and executes methods written in ASCII and stored as text files. The system was capable of handling up to four methods simultaneously. When used to carry out methods based on the APHA standard methods, the figures of merit obtained were considered satisfactory for the purpose. The total cost was under US $4600. It was concluded that the analyser is appropriate for routine use and has potential for an increased number of simultaneous methods and for enhanced capabilities if new versions of the software are developed.

Comprehensive study of the parameters influencing the detection of organotin compounds by a pulsed flame photometric detector in sewage sludge

An investigation of the operating conditions of a pulsed flame photometric detection (PFPD) system for the determination of organotin compounds (OTCs) in sewage sludge is reported. During the analyses, some spectral interferences were observed. For their elimination detector parameters such as gate delay and gate width were investigated. In addition, the applicability of three different internal standards was evaluated. Under optimised analytical conditions (gate delay 3 ms, gate width 2 ms, tripropyltin as internal standard) limits of detection (LOD) were determined. The LOD for butyltins ranged between 8 and 16 ng Sn g −1, for phenyltins around 8 ng Sn g −1 and for octyltins between 5 and 10 ng Sn g −1. Since there is no certified reference material (CRM) available for sewage sludge, the accuracy of the analytical procedure was checked by the analysis of CRM PACS-2 (marine sediment) and a spiked sludge sample. Good agreement between determined and certified values was obtained. Sewage sludge from a local wastewater treatment plant was analysed and the results compared with data from the literature.

P24 Comparison of sampling bags used for measuring breath odor

The use of sampling bags to collect breath odor has not been well established. The aim of this study was to compare and judge the quality and performance of three commercially available sampling bags. An aluminized polyethylene bag (Aluminum Bag, Tokyo Deodorant Inc., Japan), the Tedlar® bag (Tedlar® Bag, Tokyo Deodorant Inc.), and a polyester bag (Flek‐Sampler® Nioibukuro; Omi Odorair Service Co. Ltd., Japan) were assessed. A GC instrument (GC14‐B, Shimadzu, Japan) equipped with a flame photometric detection system were used to measure the concentrations of volatile sulfur compounds (VSC: hydrogen sulfide, methyl mercaptan, and dimethyl sulfide) released from the sampling bags over time. An electronic nose (e‐nose) (Odor Discrimination Analyzer FF‐1, Shimadzu, Japan) was used to assess the characteristics of the gas eluted from the inner surfaces of the bags. The GC results showed that the Tedlar® bag had the highest performance, with 73% of the VSC concentration lasting for up to 4 days. The disadvantage of this bag, however, is that the Tedlar® bag itself has an odor that disturbs the e‐nose's response and organoleptic evaluation. The polyester bags were only able to hold hydrogen sulfide for 2 h. The e‐nose's responses were strongly associated with changes in humidity within the polyester bag. The humidity was more stable within the aluminized polyethylene bag, which was able to sustain 94% of the VSC concentration for up to 24 h. In conclusion, Tedlar® bags are beneficial for GC usage. Aluminum coated polyester bags are beneficial for GC measurements, e‐nose measurements, and organoleptic judgements. Polyester bags have poor hold of VSC, but the low cost suits required disposability for the measurements of individual patients. Thus, breath odor sampling bags should be selected based on the specificity of the measuring instrument.

A Technique for Photometric Detection and Measurementof Unresolved Binary Systems

ABSTRACTA technique is described for the detection and measurement of close binary systems whose images are unresolved. The method is based on analysis of the moment‐of‐inertia tensor of the image, from which the product of the binary flux ratio and square of the angular separation may be determined. Intrinsic asymmetries of the point‐spread function are removed by comparison with the image of a reference star. Multiple exposures may be used to increase the signal‐to‐noise ratio without need of image alignment. An example is given of a simulated measurement of the dwarf carbon star system G77‐61.

Highly sensitive amperometric enzyme immunoassay for α-fetoprotein in human serum

A sandwich amperometric enzyme immunoassay with flow injection for α-fetoprotein in human serum has been developed with alkaline phosphatase as the enzyme label. p-hydroxyphenyl phosphate was used as the substrate for alkaline phosphatase. The hydrolysis product, hydroquinone, was detected by oxidative amperometry in a flow injection system. The amperometric wall jet detector was fitted with a glassy carbon working electrode held at 350 mV vs. Ag AgCl . The detection limit of hydroquinone in 30 mM borate buffer pH 9.5 was 1.2 × 10 −10 M (linearity range: 10 −9–5.12 × 10 −6 M). A detection limit for free alkaline phosphatase of 1.2 × 10 −15 M (linearity range: 10 −15–10 −13 M), or about 36 000 molecules, was observed (same borate buffer and incubations of 10 min at 25°C). These conditions were maintained for the amperometric α-fetoprotein immunoassay. For comparison purposes, a photometric detection system was set up, with p-nitrophenyl phosphate as enzyme substrate and the same pair of antibodies and incubation conditions. The detection limit for α-fetoprotein obtained by amperometry, 0.07 ng/ml (linearity range = 5–500 ng/ml), was 14 times lower than by photometry. The amperometric enzyme immunoassay correlates well with a commercial colorimetric immunoassay ( r = 0.986, slope = 0.967, n = 240).

Seasonal and regional variations in plankton community production and respiration for Chesapeake Bay

Plankton community production and respiration rates were examined at 3 stations representmg distinct regions along the estuanne gradient in the main stem of Chesapeake Bay, USA. Rates were measured as in vitro changes in oxygen concentration, as determined by Winkler titration with an automated photometric end-point detection system. At each station rates of both processes exhibited annual patterns which followed that of water temperature. There were distinct differences, however, among the 3 stations in the relative magnitude of metabolic rates measured. Annual rates of daytime net plankton community production were estimated to be 265, 1680, and 2040 g O2 n r 2 yrW1, while annual night-time plankton community respiration rates in the upper water column were estimated as 130, 1090, and 490 g 0; m 2 y r l at the upper, middle and lower Bay stations, respectively. Thus, whereas rates of net daytime production increased substantially moving downbay, highest measured rates of community respiration were, m fact, found in the middle region of the Bay. Annual cycles of production and respiration rates were significantly related to each other at the upper and middle stations, but unrelated at the lower station. Integrated estimates of net plankton community metabolism (production minus respiration) at the 3 stations exhibited seasonal patterns departing from balanced metabolism (production = respiration) during winter-spring and converging on zero net metabolism in summer-fall. During the cooler months net plankton metabolism was negative (net heterotrophic) at the upper station and positive (net autotrophic) at the middle and lower stations. Over the annual cycle, the 3 stations showed a longitudinal pattern of increasing die1 net plankton community metabolism, progressing from a net heterotrophy of -70 g O2 m 2 y r l in the turbid, upper Bay, to shghtly positive metabolism of 160 g 0; m-2 yr-I m the mid-region, to strong net autotrophy of 760 g Oa m 2 y r l in the less turbid, lower Bay. KEY WORDS' Plankton. Production Respiration. Net community metabohsm . Oxygen. Chesapeake Bay

Dual-wavelength photometry with light emitting diodes. Compensation of refractive index and turbidity effects in flow-injection analysis

An approach to compensate for both refractive index and turbidity effects is described. A light emitting diode-based dual-wavelength, double-beam (fully referenced), dual-flow-cell photometric detection system is used. The only requirement is that the analyte yields a product (that can be detected by absorbance measurement) as a result of a reaction which does not affect the optical properties of the suspended matter. The principle is mathematically established and experimentally demonstrated by determining micromolar concentrations of bromthymol blue in samples containing up to 1.5% whole milk and 60% ethanol with an absolute error of < 3.7%.

Measurements of atmospheric dimethylsulfide, hydrogen sulfide,and carbon disulfide during GTE/CITE 3

Measurements of atmospheric dimethylsulfide (DMS), hydrogen sulfide (H2S), and carbon disulfide (CS2) were made over the North and South Atlantic Ocean as part of the Global Tropospheric Experiment/Chemical Instrumentation Test and Evaluation (GTE/CITE 3) project. DMS and CS2 samples were collected and analyzed using an automated gas chromatography/flame photometric detection system with a sampling frequency of 10 min. H2S samples were collected using silver nitrate impregnated filters and analyzed by fluorescence quenching. The DMS data from both hemispheres have a bimodal distribution. Over the North Atlantic this reflects the difference between marine and continental air masses. Over the South Atlantic it may reflect differences in the sea surface source of DMS, corresponding to different air mass source regions. The median boundary layer H2S and CS2 levels were significantly higher in the northern hemisphere than the southern hemisphere, reflecting the higher frequency of samples influenced by pollutant and/or coastal emissions. Composite vertical profiles of DMS and H2S are similar to each other, and are consistent with a sea surface source. Vertical profiles of CS2 have maxima in the free troposphere, implicating a continental source. The low levels of H2S and CS2 found in the southern hemisphere constrain the role of these compounds in global budgets to significantly less than previously estimated.

Evaluation of the mla 1000 coagulation autoanalyser

The MLA Electra 1000 is a new fully automated coagulation analyser which uses a photometric detection system to perform clotting and chromogenic assays. The instrument has been evaluated for routine use in a clinical laboratory. Precision performance was determined for prothrombin time, activated partial thromboplastin time, Clauss fibrinogen assay, derived fibrinogen assay, factor VIII coagulant assay, chromogenic antithrombin III, plasminogen and protein C assays. The coefficient of variation was acceptable for all these assays, however the derived fibrinogen assay did not always correlate with that of the Clauss technique. The MLA 1000 is a versatile, easily operated instrument which yeilds reproducible results.

Isotachophoretic analyser with optioins for operational electrolyte selection and repeated analysis

A prototype of an isotachophoretic analyser with options for electrolyte selection and repeated analysis was constructed, which might be useful for the optimization of separation and the measurement of qualitative indices at different pH values for mobility and p K a evaluation. After a sample mixture had been injected into the system it was separated and pushed back to the original position, and this cycle was repeated varying the leading electrolyte. The sample was pushed back by the use of the counter flow technique. The process of repeated analysis was monitored by the use of a 32-channel UV photometric zone detection system. It was found that the flow-rate of push-back was decisively important for good reproducibility in repeated analysis. The efficiency of the apparatus was demonstrated with a model mixture of anions.

Isotachophoretic analyser equipped with a scanning ultraviolet photometric detection system

An isotachophoretic analyser with a scanning ultraviolet (UV) photometric detection system was constructed in order to study separation processes. A fused-silica capillary (I.D. 0.53 mm, O.D. 0.66 mm) was scanned over a length of 32 cm with an assembly of a UV lamp and a detector. The assembly was driven by a linear head with a stepping motor. One scanning cycle took 7.027 s and 5333 photometric signals (a UV position spectrum) were acquired. The practical resolution was 0.15 mm. The detection limit of picric acid was 20 pmol. Using the apparatus, the separation of a four-component mixture (SPADNS, monochloroacetic acid, picric acid and acetic acid) was studied using different electrolyte systems and the effect of the pH of leading electrolyte on the separation efficiency is briefly discussed.

Study of the film formation process in liquid-liquid extraction flow injection analysis

The behaviour of a thin film of an organic solvent on the walls of the extraction coil in a continuous liquid-liquid extraction flow system was studied using a computer-controlled fast-recording on-tube photometric detection system (approx. 3 ms time resolution). A single-loop injector was employed to introduce precise, reproducible volumes (Sr &lt; 2%) of one phase into the continuous stream of the other as a segmented volume standard. The film thickness Df, ranging from 1 to 20 μm for a 0.7 mm teflon tube, was calculated from the segment lengthening at a different chloroform flow rates and was found to obey a polynominal dependence on the linear flow rate, df = f(uα), where α &lt; 1.