Development of an electrochemical immunosensor for direct detection of interferon-gamma at the attomolar level

An electrochemical immunosensor for direct detection of the 15.5-kDa protein interferon-gamma (IFN-gamma) at attomolar level has been developed. Self-assembled monolayers (SAMs) of cysteine or acetylcysteine are formed on electropolished polycrystalline Au electrodes. IFN-gamma adsorbs physically to each of these SAMs. With injections of 100 mM KCl, IFN-gamma can be removed in the flow without damaging the acetylcysteine SAM. However, the cysteine SAM is affected by these KCl injections. In an on-line procedure in the flow, a specific antibody (MD-2) against IFN-gamma is covalently attached following carbodiimide/succinimide activation of the SAM. The activation of the carboxylic groups, attachment of MD-2, and deactivation of the remaining succinimide groups with ethanolamine are monitored impedimetrically at a frequency of 113 Hz, a potential of +0.2 V versus SCE, and an ac modulation amplitude of 10 mV. Plots of the real (Z') and imaginary (Z") component of the impedance versus time provide the information to control these processes. In the thermostated setup (23.0 degrees C), samples of unlabeled IFN-gamma (in phosphate buffer pH 7.4) are injected and the binding with immobilized MD-2 is monitored with ac impedance or potential-step methods. While the chronoamperometric results are rather poor, the ac impedance approach provides unsurpassed detection limits, as low as 0.02 fg mL-1 (approximately 1 aM) IFN-gamma. From a calibration curve (i.e. Z" versus the amount injected), recorded by multiple 50-microL injections of 2 pg mL(-1) of IFN-gamma, a dynamic range of 0-12 pg mL(-1) could be derived. However, when nonspecific adsorption is taken into account, which has been found to be largely reduced through injections of 100 mM KCl, a much smaller dynamic range of 0-0.14 fg mL(-1) remains. The immunosensor can be regenerated by using a sequence of potential pulses in the flow by which the SAM with attached MD-2 and bound IFN-gamma is completely removed. When the developed procedures described above are repeated, the response of the immunosensor is reproducible within 10%.     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

Disposable electrochemical sensor for determination of nitroaromatic compounds by a single-run approach

Environmental and security applications have generated major demands for effective field-deployable tools for detecting nitroaromatic compounds, such as chloramphenicol (an antibiotic), parathion (an organophosphate nerve agent), and TNT (2,4,6-trinitrotoluene, an explosive) in a fast, simple, sensitive, reliable, and cost-effective manner. We report here a single-run approach for such a purpose. The reduction potential of different nitroaromatic compounds was found to systematically shift with the substituent group at an electrochemically preanodized screen-printed carbon electrode. The preanodization treatment makes the peak sharp and hence provides a precise way to identify the substituent effect on nitroaromatic compounds. By using potential shifts as analytical characteristics of nitroaromatic compounds, a suitable internal standard can be chosen based on the criteria of well-separated peak potential and rarely found in the real sample of interest. Simply by measuring the ratio of peak currents between analytes of interest and internal standard, the analysis can be done in a single-run measurement. Both the matrix effect and the variation of electrode during the preparation process can be canceled out in this approach and thus allows for a high-precision analysis. Just by placing a 20-microL drop on a single-use amperometric sensor strip incorporating a three-electrode configuration is enough for rapid and sensitive detection of nitroaromatic compounds by square-wave voltammetry. For example, the linear detection range can be up to 100 microM with a detection limit of 0.42 microM (S/N = 3) in the detection of chloramphenicol. This approach was successfully demonstrated in real sample analysis to verify the applicability of the method. The promising performances open new possibilities for rapid determination of nitroaromatic compounds in environmental and biological samples.     

Electronic barcoding of a viral gene at the single-molecule level

A new single-molecule approach for rapid and purely electronic discrimination among similar genes is presented. Combining solid-state nanopores and γ-modified synthetic peptide nucleic acid probes, we accurately barcode genes by counting the number of probes attached to each gene and measuring their relative spacing. We illustrate our method by sensing individual genes from two highly similar human immunodeficiency virus subtypes, demonstrating feasibility of a novel, single-molecule diagnostic platform for rapid pathogen classification.     

Optical sensors for the determination of concentrated hydroxide

An optical sensor system has been developed for the determination of concentrated strong bases ([OH-] = 1-10 M). The base sensors consist of SiO2/ZrO2-organic polymer composites and doped high-pKa indicators. Films were obtained by spin-casting these composite materials on glass substrates and were used as sensor elements for the spectrometric determination of hydroxide. The hydrophilic nature of the mixed oxide SiO2/ZrO2 and its chemical stability in concentrated alkali made it attractive as support in the composites. The organic polymers in the composites either provided better mechanical stability and dye immobilization or enhanced OH- diffusion and sensor response. The composite sensors showed a relative standard deviation of less than 2%. The response time of a SiO2/ZrO2-Nafion composite (sensor 2) was short (5 s), and a small hysteresis was observed during reproducibility measurements with 1-4 M NaOH solutions. The sensors were found to be stable in 4 M NaOH during a 30-day durability test, showing a standard deviation of 3.0-4.7%. The diffusion kinetics and hysteresis performance of the sensors were also evaluated.     

Integrated inductive sensors for the detection of magnetic microparticles

In this paper, we deal with novel inductive microsensors, realized by using standard CMOS microelectronic technologies, for the detection of small amounts of magnetic beads that are placed in selected regions over the surface of the microsensor. The sensor proposed here represents a step toward the development of integrated inductive biosensors for application in the area of magnetic immunoassay where magnetic markers, carrying specific antibodies that selectively bind to the cells or molecules to be detected, are used; the measurement of the analyte concentration is therefore accomplished by determining the concentration of magnetic particles tied to it. A planar differential transformer structure is proposed here as part of the measurement strategy. The analysis, simulation, and design of the proposed device are reported, and its sensitivity to the quantity of micromagnetic beads deposited over its surface has been also demonstrated through experiments.     

Development of a microfluidic platform with an optical imaging microarray capable of attomolar target DNA detection

In this paper, DNA hybridization in a microfluidic manifold is performed using fluorescence detection on a fiber-optic microarray. The microfluidic device integrates optics, sample transport, and fluidic interconnects on a single platform. A high-density optical imaging fiber array containing oligonucleotide-labeled microspheres was developed. DNA hybridization was observed at concentrations as low as 10 aM with response times of less than 15 min at a flow rate of 1 microL/min using 50 microL of target DNA samples. The fast response times coupled with the low sample volumes and the use of a high-density, fiber-optic microarray format make this method highly advantageous. This paper describes the initial development, optimization, and integration of the microfluidic platform with imaging fiber arrays.     

Circular arrays of magnetic sensors for current measurement

A single solid-state magnetic sensor can measure a current flowing in a conductor in a noncontact way. In order to improve the accuracy of the measuring system and to reduce the crosstalk effects of other magnetic fields, circular arrays of magnetic sensors can be fabricated, still preserving low costs. In those kind of arrays, the sensing elements are assembled on a circle around the conductor. The sum of the sensor output signals is an approximation of Ampere's circulation. A quite satisfactory crosstalk effects reduction can be thus achieved. More sophisticated algorithms that combine sensor output signals are necessary in order to further reduce crosstalk effects. This paper presents an algorithm that is able to calculate the intensity of a dc current flowing in a rectangular bus-bar, in the presence of crosstalk fields     

Disposable biosensor based on a hemoglobin colloidal gold-modified screen-printed electrode for determination of hydrogen peroxide

A disposable reagentless hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin immobilized on a colloidal gold-modified screen-printed carbon electrode (Hb-Au-SPCE) was proposed. The electrochemical behavior of immobilized Hb at a SPCE was studied for the first time. The electrode reaction of immobilized Hb showed a surface-controlled process with an electron transfer rate constant of (0.40 /spl plusmn/ 0.02) s/sup -1/ determined in the scan rate range from 25 to 200 mV s/sup -1/. The Hb-Au-SPCE exhibited an electrocatalytic activity toward the reduction of hydrogen peroxide with a K/sub M//sup app/ value of 1.8 mM, which was allowed to be used as a disposable sensor for determination of hydrogen peroxide with a linear range from 1.0 /spl times/ 10/sup -5/ M to 3.2 /spl times/ 10/sup -4/ M, a detection limit of 5.5 /spl times/ 10/sup -6/ M at 3/spl sigma/, a high sensitivity, fast response, and good selectivity, accuracy, and reproducibility. The disposable reagentless sensor was stable, low cost, and simple to use for detection of hydrogen peroxide in real samples.     

Multisegment nanowire sensors for the detection of DNA molecules

We describe a novel application for detecting specific single strand DNA sequences using multisegment nanowires via a straightforward surface functionalization method. Nanowires comprising CdTe-Au-CdTe segments are fabricated using electrochemical deposition, and electrical characterization indicates a p-type behavior for the multisegment nanostructures, in a back-to-back Schottky diode configuration. Such nanostructures modified with thiol-terminated probe DNA fragments could function as high fidelity sensors for biomolecules at very low concentration. The gold segment is utilized for functionalization and binding of single strand DNA (ssDNA) fragments while the CdTe segments at both ends serve to modulate the equilibrium Fermi level of the heterojunction device upon hybridization of the complementary DNA fragments (cDNA) to the ssDNA over the Au segment. Employing such multisegment nanowires could lead to the fabrication more sophisticated and high multispecificity biosensors via selective functionalization of individual segments for biowarfare sensing and medical diagnostics applications.     

Biomimetic sensors of molecularly-imprinted polymers for chlorpromazine determination

A new man-tailored biomimetic sensor for Chlorpromazine host-guest interactions and potentiometric transduction is presented. The artificial host was imprinted within methacrylic acid, 2-vinyl pyridine and 2-acrylamido-2-methyl-1-propanesulfonic acid based polymers. Molecularly imprinted particles were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. Slopes and detection limits ranged 51–67 mV/decade and 0.46–3.9 μg/mL, respectively, in steady state conditions. Sensors were independent from the pH of test solutions within 2.0–5.5. Good selectivity was observed towards oxytetracycline, doxytetracycline, ciprofloxacin, enrofloxacin, nalidixic acid, sulfadiazine, trimethoprim, glycine, hydroxylamine, cysteine and creatinine. Analytical features in flowing media were evaluated on a double-channel manifold, with a carrier solution of 5.0 × 10^?2 mol/L phosphate buffer. Near-Nernstian response was observed over the concentration range 1.0 × 10^?4 to 1.0 × 10^?2 mol/L. Average slopes were about 48 mV/decade. The sensors were successfully applied to field monitoring of CPZ in fish samples, offering the advantages of simplicity, accuracy, automation feasibility and applicability to complex samples.     

Biomolecular sensors for neurotransmitter determination: electrochemical immobilization of glutamate oxidase at microelectrodes in a poly(o-phenylenediamine) film

The adsorption of glutamate oxidase onto 25 m and 10 m platinum microelectrodes followed by immobilization in an electrochemically polymerized non-conducting polymer, poly(o-phenylenediamine), is described as a method of fabricating an enzyme electrode for the amperometric determination of glutamate. The response of the enzyme electrodes were found to be highly reproducible with a linear dynamic range upto approximately 15 mmol dm^–3. The response of the 25 m and 10 m enzyme electrodes to glutamate were analysed using an established kinetic model and the potential application of the sensor for the study of neurotransmitter dynamics was investigated. The sensor was stable over a period of 30 days and the polymeric film was found to reduce interference from the electroactive compounds, uric acid and ascorbic acid.     

Biomolecular sensors for neurotransmitter determination: electrochemical immobilization of glutamate oxidase at microelectrodes in a poly(o-phenylenediamine) film

The adsorption of glutamate oxidase onto 25 Μm and 10 Μm platinum microelectrodes followed by immobilization in an electrochemically polymerized non-conducting polymer, poly(o-phenylenediamine), is described as a method of fabricating an enzyme electrode for the amperometric determination of glutamate. The response of the enzyme electrodes were found to be highly reproducible with a linear dynamic range upto approximately 15 mmol dm^?3. The response of the 25 Μm and 10 Μm enzyme electrodes to glutamate were analysed using an established kinetic model and the potential application of the sensor for the study of neurotransmitter dynamics was investigated. The sensor was stable over a period of 30 days and the polymeric film was found to reduce interference from the electroactive compounds, uric acid and ascorbic acid.     

Design of inductive sensors for magnetic testing of steel ropes

The design and operating principles of four inductive sensors for magnetic testing of steel ropes are presented. The magnetic concentrators can maintain the same shape as in Hall-effect leakage flux sensors, but the output signals of the inductive sensors are quite different and depend on the speed of testing. Although the inductive sensors are not as versatile as Hall-effect sensors, they are simpler in operation and can still find applications, especially in the initial and middle stages of the deterioration of the rope.     

Design of Hall-effect sensors for magnetic testing of steel ropes

The design and operating principles of four new Hall-effect sensors for magnetic testing of steel ropes are presented. The radial or tangential component of the magnetic leakage flux can be measured efficiently depending on the shape of the concentrators (rings or sleeves). The output signal can be modified by using multiple constructions of the basic sensors. Different sensors can be used depending on the predominant character of the wear of the rope.     

Investigation of cryogenic level sensors for LN2 and LOX

Investigations of two different types of cryogenic level sensors (capacitance and High Temperature Superconductor (HTS) for level measurement of liquid nitrogen (LN₂) and liquid oxygen (LOX) are presented here. They were tested for an active length of 400 mm in LOX and LN₂. A discrete diode array level sensor was used as a primary standard for calibrating these sensors. Comparative studies on linearity, sensitivity and other parameters at the operating temperatures are presented.     

Accurate determination of specific heat at high temperatures using the flash diffusivity method

The flash diffusivity method can be extended, very simply, to measuring simultaneously thermal diffusivity and specific heat and thus obtaining the thermal conductivity directly. This was accomplished by determining the amount of heat absorbed by a sample with a well-known specific heat and then using this to determine the specific heat of any other sample. The key to using this technique was to have identically reproducible surfaces on the standard and the unknowns. This was achieved earlier by sputtering the surfaces of the samples with a thin layer of graphite. However, the accuracy in determining the specific heat was within ±10% and there was considerable scatter in the data. Several improvements in the technique have been made which have improved the accuracy to ±3% and increased the precision. The most important of these changes has been the introduction of a method enabling the samples to be placed in exactly the same position in front of the light source. Also, the control of the thickness and the application of the graphite coating have turned out to be very important. A comparison of specific heats obtained with this improved technique and with results obtained using other techniques has been made for two materials.Paper presented at the Tenth Symposium on Thermophysical Properties, June 20–23, 1988, Gaithersburg, Maryland, U.S.A.     

Losses in composite superconductors at a high level of magnetic field excitation: Part 2

Losses in a round composite superconductor are discussed. The calculated results are in good agreement with experimental data obtained. An analysis is provided of the effect of the collective interaction between turns in the winding at a high level of excitation, ie, under conditions where the saturated zone occupies a considerable part of the composite volume.