Electrochemical detection of high-sensitivity CRP inside a microfluidic device by numerical and experimental studies.

The concentration of C-reactive protein (CRP), a classic acute phase plasma protein, increases rapidly in response to tissue infection or inflammation, especially in cases of cardiovascular disease and stroke. Thus, highly sensitive monitoring of the CRP concentration plays a pivotal role in detecting these diseases. Many researchers have studied methods for the detection of CRP concentrations such as optical, mechanical, and electrochemical techniques inside microfluidic devices. While significant progress has been made towards improving the resolution and sensitivity of detection, only a few studies have systematically analyzed the CRP concentration using both numerical and experimental approaches. Specifically, systematic analyses of the electrochemical detection of high-sensitivity CRP (hsCRP) using an enzyme-linked immunosorbant assay (ELISA) inside a microfluidic device have never been conducted. In this paper, we systematically analyzed the electrochemical detection of CRP modified through the attachment of an alkaline phosphatase (ALP-labeled CRP) using ELISA inside a chip. For this analysis, we developed a model based on antigen-antibody binding kinetics theory for the numerical quantification of the CRP concentration. We also experimentally measured the current value corresponding to the ALP-labeled CRP concentration inside the microfluidic chip. The measured value closely matched the calculated value obtained by numerical simulation using the developed model. Through this comparison, we validated the numerical simulation methods, and the calculated and measured values. Lastly, we examined the effects of various microfluidic parameters on electrochemical detection of the ALP-labeled CRP concentration using numerical simulations. The results of these simulations provide insight into the microfluidic electrochemical reactions used for protein detection. Furthermore, the results described in this study should be useful for the design and optimization of electrochemical immunoassay chips for the detection of target proteins.

Highly sensitive potentiometric strip test for detecting high charge density impurities in heparin.

Contamination of heparin with oversulfated chondroitin sulfate (OSCS) became a matter of grave concern in the medical field after many fatal responses to OSCS tainted heparin products occurred during the 2007-2008 period. Even though standard lab-based analytical techniques such as nuclear magnetic resonance (NMR) and strong anion-exchange high performance liquid chromatography (SAX-HPLC) have proven useful for monitoring the OSCS content in heparin products, an easy-to-use, quick, portable, and cost-efficient method is still needed for on-site monitoring during and after the heparin production. In this report, a disposable strip-type electrochemical polyion sensor is described for detection of low levels of OSCS contamination in heparin. A magnetic actuator is incorporated into this simple electrode-based microfluidic device in order to create the mixing effect necessary to achieve equilibrium potential changes of the sensor within a microfluidic channel. The planar membrane electrode detector within the sample channel is prepared with a tridodecylmethylammonium chloride (TDMAC)-doped poly(vinyl chloride) (PVC) membrane essentially equivalent to previously reported polyanion-sensitive electrodes. When the concentration of heparin applied to the single-use strip device is 57 mg/mL (in only 20 µL of sample), the same concentration recommended in the NMR analysis protocol for detecting OSCS in heparin, the detection limit is 0.005 wt % of OSCS, which is ca. 20 times lower than the reported detection limit of the NMR method.

Selective electrochemical sensing of glycated hemoglobin (HbA1c) on thiophene-3-boronic acid self-assembled monolayer covered gold electrodes.

We report a novel concept of sensing glycated hemoglobin, HbA 1c, which is now the most important index for a long-term average blood glucose level, by first selectively immobilizing it on the thiophene-3-boronic acid (T3BA) self-assembled monolayer (SAM)-covered gold electrode by a selective chemical reaction with boronic acid. HbA 1c thus immobilized is then detected by the label-free electrochemical impedance spectroscopic (EIS) measurements with a redox probe, an equimolar mixture of K 3Fe(CN) 6 and K 4Fe(CN) 6, present. The rate of charge transfer between the electrode and the redox probe is shown to be modulated by the amount of HbA 1c in the matrix hemoglobin solution due to the blocking effect caused by the binding of HbA 1c with boronic acid. Both the formation of a well-defined T3BA-SAM on the gold surface and the chemical binding of its boronic acid with HbA 1c in solution were confirmed by quartz crystal microbalance, atomic force microscopy, and EIS experiments.

A rapid competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) test strip for microcystin-LR (MCLR) determination.

A competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) is described for the determination of microcystin-LR (MCLR) using a double-sided microporous gold electrode in cartridge-type cells. A gold film sputtered on one side of porous nylon membrane constitutes a working electrode, while another gold film formed on the opposite side serves as a pseudo reference electrode. After immobilizing MCLR antibody on working electrode by physical adsorption, the double-sided electrode was placed simply in a diffusion U-type or within a dry strip-type cell with a conjugate pad pre-loaded with a glucose oxidase labeled MCLR (GOx-MCLR) on working electrode side. Assays were performed in two steps: an MCLR-containing sample mixed with a known amount of GOx-MCLR conjugate either in buffer solution or in pre-loaded dry pad was incubated for an appropriate period (about 10 min) to induce competitive reaction with an immobilized anti-MCLR antibody on working electrode, and a fixed concentration of glucose solution (substrate) was then added to the backside of the working electrode. Due to the competitive nature of the assay, enzymatically generated product, hydrogen peroxide (H2O2), was detected at the working gold electrode (at +800 mV versus Au) by oxidation, and the magnitude of amperometric current was inversely proportional to the concentration of MCLR in the sample. The response time after substrate addition was about 30s. Mean recovery of MCLR added to tap water was 93.5%, with a coefficient of variation (CV) of 6.6%. The proposed competitive NEEIA system is in general comparable to existing heterogeneous enzyme immunoassays with a similar detection limit (100 pg/mL MCLR), and suitable for developing a disposable type biosensor for on-site monitoring of environment.

Electrochemical characterization of biosensor based on nitrite reductase and methyl viologen co-immobilized glassy carbon electrode.

Nitrite reductase (NiR, nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1) and methyl viologen (MV) were co-immobilized on glassy carbon electrode (GCE, d=3 mm) by polymer entrapment, and the electrode was tested as an electrochemical biosensor for amperometric determination of nitrite. The immobilization was performed by sequential loading and drying of a homogeneous mixture of poly(vinyl alcohol) (PVA), NiR and MV, followed by poly(allylamine hydrochloride) (PAH) solution, and finally hydrophilic polyurethane (HPU) dissolved in chloroform. The positively charged PAH layer could effectively keep immobilized cationic MV from diffusing through the membrane, holding mediator tightly near or on the electrode surface. The working principle of the biosensor was based on MV mediated electron transfer between electrode and immobilized NiR. The response time (t(90%)) of the biosensor was about 20 s and sensitivity was 11.8 nA/ microM (2.5 mU NiR) with linear response range of 1.5-260 microM (r(2)=0.996) and detection limit of 1.5 microM (S/N=3). Lineweaver-Burk plot showed that Michaelis-Menten constant (K(m,app)) was about 770 microM. The biosensor showed durable storage stability for 24 days (stored in ambient air at room temperature) retaining 80% of its initial activity, and showed satisfactory reproducibility (relative standard deviation (R.S.D.)=3.8%, n=9). Interference study showed that chlorate, chloride, sulfite, sulfate did not interfere with the nitrite determination, however, nitrate interfered with the determination with relative sensitivity of 38% (ratio of sensitivity for nitrate to that for nitrite). In addition to the full characterization of the biosensor, kinetic study was also conducted in solution and the homogeneous rate constant (k(2)) between NiR and MV were determined by chronoamperometry to be 5.8 x 10(5) M(-1) s(-1).

A correction method using a support vector machine to minimize hematocrit interference in blood glucose measurements.

Point-of-care testing glucose meters are widely used, important tools for determining the blood glucose levels of people with diabetes, patients in intensive care units, pregnant women, and newborn infants. However, a number of studies have concluded that a change in hematocrit (Hct) levels can seriously affect the accuracy of glucose measurements. The aim of this study was to develop an algorithm for glucose calculation with improved accuracy using the Hct compensation method that minimizes the effects of Hct on glucose measurements. The glucose concentrations in this study were calculated with an adaptive calibration curve using linear fitting prediction and a support vector machine, which minimized the bias in the glucose concentrations caused by the Hct interference. This was followed by an evaluation of performance according to the international organization for standardization (ISO) 15197:2013 based on bias with respect to the reference method, the coefficient of variation, and the valid blood samples/total blood samples within the ±20% and 15% error grids. Chronoamperometry was performed to verify the effect of Hct variation and to compare the proposed method. As a result, the average coefficients of variation for chronoamperometry and the Hct compensation method were 2.43% and 3.71%, respectively, while the average biases (%) for these methods were 12.08% and 5.69%, respectively. The results of chronoamperometry demonstrated that a decrease in Hct levels increases glucose concentrations, whereas an increase in Hct levels reduces glucose concentrations. Finally, the proposed method has improved the accuracy of glucose measurements compared to existing chronoamperometry methods.

Glass nanopore-based ion-selective electrodes.

Glass nanopore-based all-solid-state ion-selective electrodes (ISEs) have been developed to probe the distribution of ionic species at micro- or submicrometer-length scales, e.g., mapping of ion flux through micrometer-sized pores. The all-solid-state ISE was fabricated by sealing a conically etched platinum wire (d = 25-microm; radius of etched tip <10 nm) into a soda lime glass capillary. A Pt disk was exposed by gentle polishing the glass and the disk etched to form a conical pore of submicrometer dimension (radius < approximately 500 nm; depth < approximately 30 microm). Ag was electroplated on the Pt electrode in the pore and gently chloridated to obtain a AgCl/Ag layer within the pore. The AgCl/Ag layer-coated ISE was used as a highly selective Cl- probe in scanning electrochemical microscope experiments to map the ion flux through a micropore. The same ISE was also used as the base transducer of the neutral carrier-doped solvent polymeric membrane. The optimized polymer membranes used for the glass nanopore-based all-solid-state ISE require a higher ratio of plasticizer/polymer (9/1) compared to those for conventional ISE (2/1). An ISE based on deposition of an IrO2 layer at the base of a glass nanopore electrode exhibited a highly sensitive response (79.7 +/- 2.3 mV/pH) to variations in pH and could be used for approximately 3 weeks.

Cation selectivity of ionophores based on tripodal thiazole derivatives on benzene scaffold.

The synthesis and potentiometric evaluation of new 1,3,5-tris(thiazolylcarbethoxy)-2,4,6-trimethylbenzene (3), 1,3,5-tris(thiazolylhydroxy)-2,4,6-trimethylbenzene (4), 1,3,5-tris(thiazolylmethyl)-2,4,6-trimethylbenzene (5), and 1,3,5-tris(thiazolylphenyl)-2,4,6-trimethylbenzene (6), toward mono and divalent cations under various pH conditions are outlined. The ion-selective properties of the newly synthesized compounds were studied by measuring the potentiometric responses of the 3-, 4-, 5-, and 6-based membrane electrodes to alkali metal, alkaline earth metal, ammonium, and transition metal ions, under various pH conditions. The 3-based electrode exhibited a Nernstian response to ammonium and potassium under alkaline pH conditions, while the other three electrodes showed a poor potentiometric performance. All electrodes showed substantial responses to silver ion under acidic condition, but there was almost nil response to other transition metal ions (Fe(2+), Co(2+), Zn(2+), Ni(2+), Pb(2+), Cd(2+), Cu(2+) and Hg(2+)). The 3- and 5-based electrodes resulted in near Nernstian responses (51.3mV and 59.5mV/pAg(+), respectively) with low detection limits (approximately 100ppt), while the 4- and 6-based ones showed sub-Nernstian below 40mV/pAg(+). The results were interpreted with semi-empirically modeled structures.

Electrochemical determination of nitrate with nitrate reductase-immobilized electrodes under ambient air.

Nitrate monitoring biosensors were prepared by immobilizing nitrate reductase derived from yeast on a glassy carbon electrode (GCE, d = 3 mm) or screen-printed carbon paste electrode (SPCE, d = 3 mm) using a polymer (poly(vinyl alcohol)) entrapment method. The sensor could directly determine the nitrate in an unpurged aqueous solution with the aid of an appropriate oxygen scavenger: the nitrate reduction reaction driven by the enzyme and an electron-transfer mediator, methyl viologen, at -0.85 V (GCE vs Ag/AgCl) or at -0.90 V (SPCE vs Ag/AgCl) exhibited no oxygen interference in a sulfite-added solution. The electroanalytical properties of optimized biosensors were measured: the sensitivity, linear response range, and detection limit of the sensors based on GCE were 7.3 nA/microM, 15-300 microM (r2 = 0.995), and 4.1 microM (S/N = 3), respectively, and those of SPCE were 5.5 nA/microM, 15-250 microM (r2 = 0.996), and 5.5 microM (S/N = 3), respectively. The disposable SPCE-based biosensor with a built-in well- or capillary-type sample cell provided high sensor-to-sensor reproducibility (RSD < 3.4% below 250 microM) and could be used more than one month in normal room-temperature storage condition. The utility of the proposed sensor system was demonstrated by determining nitrate in real samples.

Silver(I) ion selective ionophores containing dithiocarbamoyl moieties on steroid backbone.

Tweezer-type and non-tweezer-type ionophores containing dithiocarbamoyl groups on a 7-deoxycholic amide or cholane derivatives were designed and synthesized. Potentiometric evaluation of the poly(vinyl chloride) (PVC) membranes containing those deoxycholic amides/cholanes linked with tweezer-type dithiocarbamoyl moieties showed excellent affinity and selectivity to silver(I) ion over alkali, alkaline earth and other transition metal cations. On the other hand, deoxycholic amides/cholanes substituted with one dithiocarbamoyl group, i.e., non-tweezer-type ionophores, resulted in relatively poor potentiometric sensitivity and detection limits. The enhanced potentiometric properties of newly synthesized tweezer-type dithiocarbamoyl containing ionophores have been further improved by employing silver ion complexing reagent in the internal reference solution, which resulted in greatly reduced detection limit ( approximately 100ppt) for the electrodes based on them.

All-solid-state carbonate-selective electrode based on a molecular tweezer-type neutral carrier with solvent-soluble conducting polymer solid contact.

Carbonate-selective membranes were prepared by incorporating a molecular tweezer-type carbonate-selective neutral carrier [N,N-dioctyl-3alpha,12alpha-bis(4-trifluoroacetylbenzyloxy)-5beta-cholan-24-amide] into a room temperature vulcanizing-type silicone rubber (3140 RTV-SR) matrix, and deposited on the planar-type electrodes (Pt containing Ag/AgCl electrodes formed on a ceramic plate) with and without an intermediary conducting polymer layer. Two types of solvent-soluble conducting polymers [poly(1-hexyl-3,4-dimethyl-2,5-pyrrolylene) or poly(3-octylthiophene-2,5-diyl)] have been examined as the solid contact material. Potentiometric properties of the resultant all-solid-state electrodes were evaluated in terms of their carbonate selectivity, response slope, potential stability and reproducibility. The sensitivity and carbonate selectivity of the SR membrane-based all-solid-state electrodes with conducting polymer solid contact were comparable to those of conventional electrodes. Experimental results also showed that the intermediary conducting polymer layer used in the all-solid-state electrodes greatly reduces the interference from dissolved oxygen.

Characterization of epoxy resin-based anion-responsive polymers: applicability to chloride sensing in physiological samples.

The potentiometric properties of an epoxy resin-based anion-responsive polymer and its feasibility as a chloride sensing membrane are described. The response mechanism of the epoxy resin-based membrane is studied by varying the types and contents of epoxy components (e.g., resin, hardener, diluent, and plasticizer). It is found that epoxy resins cured with polyamine-type hardeners have excellent characteristics as chloride-sensing membranes, in terms of their selectivity, stability, sensor lifetime, and adhesion for constructing an all-solid-state sensing device. In addition, effects of the diluent or plasticizer added in an epoxy resin/hardener system on the potentiometric behaviors are investigated. To demonstrate the practical analytical utility of the epoxy resin-based electrodes, chloride levels in various controls and biological specimens (e.g., human sera and whole blood) are determined with a flow-cell system.

Tweezer-type neutral carrier-based calcium-selective membrane electrode with drastically reduced anionic interference.

A new calcium-selective ionophore N,N-dioctyl-3alpha,12alpha-bis(N-heptyl-N-methylcarbamoyl-methoxyacetamidoacetoxy)-5beta-cholan-24-amide (denoted as BACA), was synthesized, and its potentiometric performance has been evaluated in comparison with that of the best known calcium-selective neutral carriers, ETH 129 and ETH 1001. The 1H NMR spectra of BACA titrated with Ca(SCN)2 suggest that BACA forms a 1:1 complex, tweezing a calcium ion between the two parallel diamide groups substituted on a rigid bile acid frame. The calcium-selective membrane based on BACA was less selective to calcium (log K(Ca2+ j)POT = -4.2, -4.2, -4.6, and -4.8, respectively, for j = Mg2+, Li+, Na+, and K+) than those based on ETH 129 (log K(Ca2+ j)POT = -4.4, -4.3, -5.4, and -5.4, respectively, in the same order) and ETH 1001 (log K(Ca2+ j)POT = -4.4, -4.4, -5.4, and -5.4), implying that BACA forms a weaker calcium complex than the other two ETH compounds. In our experimental conditions, potentiometrically determined complex formation constants of calcium-selective neutral carriers (log beta(Ca2+ L)) were 15.2, 14.0, and 8.6 for ETH 129, ETH 1001, and BACA, respectively. A slightly reduced calcium selectivity of BACA, however, affects the anionic interference-free calcium-selective membrane; the BACA-based membrane exhibits a Nernstian response up to 10(-1) M Ca2+ in the presence of lipophilic anions (e.g., SCN-, ClO4-, salicylate, and I-) and anionic surfactant, whereas the ETH 129- and ETH 1001-based ones suffer from serious anionic interference showing a curvature or leveled off response over about 10(-4) M. It was demonstrated that such a trade off does not affect the analytical performance of BACA-based electrode in most applications, including clinical analysis.

Determination of oceanic carbon dioxide using a carbonate-selective electrode.

Potentiometric properties of the PVC membrane-based electrodes prepared with molecular tweezer-type neutral carriers, 3,12-bis(TFAB)CA and deoxy-3,12-bis(TFAB)CA, and trifluoroacetyl-p-decylbenzene (TFADB) were measured in buffered electrolytes (0.1 M Tris-H2SO4, pH 8.6 and 8.0) and artificial seawater. It was observed that the deoxy-3,12-bis(TFAB)CA-based electrode provides greatly enhanced carbonate selectivity over chloride (log K(CO3(2-), Cl-)POT approximately -6) and other minor anions present in seawater. Thus, we explored the possibility of applying this new carbonate-selective electrode for direct determination of oceanic carbon dioxide. The total carbon dioxide (TCO2) level in surface Yellow Sea water was determined with the deoxy-3,12-bis(TFAB)CA-based electrode, Severinghaus-type CO2 gas sensor, and the traditional potentiometric titration methods. The results showed that the carbonate-selective electrode provides accurate oceanic TCO2 determination comparable to that obtainable with the other two methods. The analytical procedure based on a carbonate-selective electrode is clearly advantageous over other conventional methods: it does not require any sample pretreatment and extra reagents other than the standard calibration solutions, while providing the measured results directly and immediately.

Synthesis and characterization of 7-deoxycholic amide backbone-based silver(I) ionophores.

Four ionophores containing bipyridyl groups on a 7-deoxycholic amide derivative scaffold were designed and synthesized. Potentiometric evaluation of the Poly(vinyl chloride) (PVC) membranes containing those deoxycholic amides bearing bipyridyl moieties with a short linkage showed good affinity to silver(I) ion over alkali, alkaline earth and other transition metal cations. However, two bipyridyl groups flexibly linked to the deoxycholic frame through a long linkage do not result in appreciable potentiometric responses.

Ion-selective electrodes based on molecular tweezer-type neutral carriers.

Potentiometric properties of cholic and deoxycholic acid derivatives substituted with various ion-recognizing moieties, such as dithiocarbamate, bipyridyl, glycolic and malonic diamides, urea and thiourea, and trifluoroacetophenons (TFAP), have been studied using solvent polymeric membranes. The dithiocarbamate and bipyridyl group containing ionophores exhibit high silver ion selectivity. The cholic acid derivatized with glycolic diamides exhibited high calcium selectivity, but its complex formulation constant was 10(5) times smaller than that of ETH 1001. The reduced calcium binding ability of the glycolic diamide-substituted ionophore was advantageous for eliminating anionic interference. The bi- or tripodal malonic diamide-substituted ionophores exhibited substantially increased magnesium selectivity. Anion-selective ionophores have been designed by substituting urea and thiourea group containing chains to the hydroxyl linkers of chenodeoxycholic acid frames; their selectivity closely followed the sequence of Hoffmeister series, except the unusually large response of the thiourea-substituted ionophore to sulfate. The most successful examples of cholic or deoxycholic acid frame-based ionophores are those functionalized with two carbonate-selective TFAP groups: bipodal TFAP groups behaves like a tweezers for the incoming carbonate, and exhibit analytically interference free and quantitative responses to the carbonate in serum and seawater samples.