Microfluidic biosensor based on an array of hydrogel-entrapped enzymes

Here we show that a microfluidic sensor based on an array of hydrogel-entrapped enzymes can be used to simultaneously detect different concentrations of the same analyte (glucose) or multiple analytes (glucose and galactose) in real time. The concentration of paraoxon, an acetylcholine esterase inhibitor, can be quantified using the same approach. The hydrogel micropatch arrays and the microfluidic systems are easy to fabricate, and the hydrogels provide a convenient, biocompatible matrix for the enzymes. Isolation of the micropatches within different microfluidic channels eliminates the possibility of cross talk between enzymes.     

Preparation of polyaniline nanotubes array based on anodic aluminum oxide template

In this article, the highly ordered polyaniline (PANI) nanotubes array was prepared by in situ polymerization using anodic aluminum oxide (AAO) as template. Polymerization of aniline was confined in the one-dimensional nanochannel of AAO template. The aniline was adsorbed and polymerized preferentially on the pore walls of template. The structure of PANI nanotubes array was characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), selected area electron diffraction (SAED) and dynamic force microscope (DFM). The results show that PANI nanotubes are synthesized successfully in the nanopores of template, the diameter and length of PANI nanotubes are closed to the pore diameter and thickness of AAO template, respectively, the arrangement of PANI nanotubes is very regular and uniform, the crystal form of PANI nanotubes is hexagonal, different from pseudo-orthorhombic crystal form of PANI bulk sample, and cell parameters a and b are 0.5008 nm. The change of crystal form is due to the confinement of AAO template, which makes the molecular chain of PANI arrange more ordered.     

Ammonia gas detection based on polyaniline nanofibers coated on interdigitated array electrodes

Ammonia gas sensors were fabricated from polyaniline nanofibers, which were synthesized by a simple dilute polymerization method without external template. The films of polyaniline nanofibers were deposited on interdigitated array electrodes by drop casting method. The ammonia gas sensing mechanism arises from the deprotonation process of acid doped polyaniline. The sensors exhibited the sensitivity of 1.06 and response time of 10 s for 50 ppm of ammonia gas. Such high sensitivity and fast response are attributed to the large surface-to-volume ratio and interconnected network structures. These results suggest that polyaniline nanofibers are promising materials for ammonia gas sensors with high performance.     

Electrochemical biosensor array for the identification of microorganisms based on lectin-lipopolysaccharide recognition

Rapid identification of bacterial strains remains a well-known problem in applied medicine and, for viable pathogens, is an important diagnostic goal. We have investigated an electrochemical biosensor array, in which transduction is based on respiratory cycle activity measurements, where the microorganism's native respiratory chain is interrupted with non-native external oxidants. The selective biochemical recognition agents employed in this study are lectins that, once immobilized, recognize and bind to cell surface lipopolysaccharides. Porous membranes with different surface properties were examined as potential immobilization supports for these lectins. Optimizations performed using concanavalin A and E. coli JM105 show that immobilization methods involving pre-activated membranes significantly reduce the time required to create a functional lectin layer on the membrane surface. Overall, we found general agreement between agglutination test results and the electrochemical assessment of lectin-cell binding. Chronocoulometric measurements were made for cells captured on lectin-modified Immunodyne ABC membranes physically affixed to Pt working electrodes. This lectin-based sensor array was exposed to viable cells of gram-negative and gram-positive bacteria as well as yeast, and chronocoulometric measurements were used to generate a pattern of responses for each organism toward each lectin. Principal component analysis was used to classify the chronocoulometric results for the different microbial strains. With this new method, six microbial species (Baccilus cereus, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Enterobacter aerogenes, Saccharomyces cerevisiae) were readily distinguished.     

Detection of anticancer drug tamoxifen using biosensor based on polyaniline probe modified with horseradish peroxidase

Amperometric biosensor based on horseradish peroxidase immobilized via glutaraldehyde on the polyaniline modified platinum electrode shows evidenced promising characteristics in detecting anticancer drug tamoxifen. The sensor was fabricated simply by adsorbing horseradish peroxidase enzyme on the electrode surface for which Cyclic Voltammetry was used to monitor the electro-catalytic reduction of tamoxifen under diffusion-adsorption controlled conditions. Fourier Transform Infrared Spectroscopy, Cyclic Voltammetry and Electrochemical Impedance Spectroscopic techniques are used to characterize the electrochemical interfacial properties of surface modified electrodes. The first-hand effort on modified biosensor within Platinum/Polyaniline/Horseradish peroxidase biosensor system has demonstrated excellent electro-analytical properties with biosensor sensitivity of 1.6 μA ng mL^? 1. The optimum limit of detection and limit of quantification are 0.07 ng mL^? 1 and 0.29 ng mL^? 1 respectively for the determination of anticancer drug tamoxifen. It is felt that the present study will help in improving our knowledge of cost-effective quantitative determination of tamoxifen in metabolized biological fluids and other pharmaceutical formulations.     

Amperometric biosensor based on carbon nanotubes coated with polyaniline/dendrimer-encapsulated Pt nanoparticles for glucose detection

A novel amperometric glucose biosensor based on the nanocomposites of multi-wall carbon nanotubes (CNT) coated with polyaniline (PANI) and dendrimer-encapsulated Pt nanoparticles (Pt-DENs) is prepared. CNT coated with protonated PANI is in situ synthesized and Pt-DENs is absorbed on PANI/CNT composite surface by self-assembly method. Then Glucose oxidase (GOx) is crosslink-immobilizated onto Pt-DENs/PANI/CNT composite film. The results show that the fabricated GOx/Pt-DENs/PANI/CNT electrode exhibits excellent response performance to glucose, such as low detection limit (0.5 µM), wide linear range (1 µM–12 mM), short response time (about 5 s), high sensitivity (42.0 µA mM^? 1 cm^? 2) and stability (83% remains after 3 weeks).     

A novel urea biosensor based on zirconia

Electrochemically deposited biocompatible zirconia (ZrO₂) film on gold coated glass electrodes has been utilized for the fabrication of urea biosensor. The prepared ZrO₂ films and bioelectrodes have been characterized using Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and electrochemical techniques, respectively. The urea biosensor, fabricated by immobilizing mixed enzyme [urease (Urs) and glutamate dehydrogenase (GLDH)] on this nanobiomaterial, shows linearity up to 40 mg dL^− 1 of analyte (urea) and sensitivity of 0.071 μA/(mM cm^− 2) with stability up to 4 months when stored at 4 °C. The low value of Michaelis-Menten constant (K_m) estimated using Hans plot as 0.5 mM indicates enhancement in the affinity and/or activity of enzyme attached to this nanostructured biocompatible matrix.     

Amperometric glucose biosensor based on in situ electropolymerized polyaniline/poly(acrylonitrile-co-acrylic acid) composite film

A new type of in situ electropolymerization was used for electrochemical biosensor design. The biologic film was prepared by in situ electropolymerization of aniline into microporous poly(acrylonitrile-co-acrylic acid)-coated platinum electrode in the presence of glucose oxidase. The results of EIS and SEM indicated the successful immobilization for enzyme in the composite polymer film. The novel glucose biosensor exhibited good selectivity and operational stability, which showed no apparent loss of activity after 40 consecutive measurements and intermittent usage for 45 days with storage in a phosphate buffer solution at 4°C. In addition, optimization of the biosensor construction as well as effects of applied potential, pH value of solution, temperature and common interfering compounds on the amperometric response of the sensor were investigated and discussed.     

聚苯胺纳米纤维的合成及其在生物传感器中的应用

采用三嵌段共聚物P123(EO20PO70EO20)合成聚苯胺纳米纤维。利用粉末X射线衍射、透射电子显微镜、扫描电子显微镜、紫外光谱、红外光谱、热重分析等手段对产品的形态、组成和光学性质进行了表征。另外,将聚苯胺固定到玻碳电极表面构建了生物传感器。聚苯胺修饰电极对抗坏血酸的氧化显示了高的催化活性。讨论了聚苯胺纳米纤维可能的形成机理。     

A microfluidic paper-based electrochemical biosensor array for multiplexed detection of metabolic biomarkers

Abstract

Paper-based microfluidic devices have emerged as simple yet powerful platforms for performing low-cost analytical tests. This paper reports a microfluidic paper-based electrochemical biosensor array for multiplexed detection of physiologically relevant metabolic biomarkers. Different from existing paper-based electrochemical devices, our device includes an array of eight electrochemical sensors and utilizes a handheld custom-made electrochemical reader (potentiostat) for signal readout. The biosensor array can detect several analytes in a sample solution and produce multiple measurements for each analyte from a single run. Using the device, we demonstrate simultaneous detection of glucose, lactate and uric acid in urine, with analytical performance comparable to that of the existing commercial and paper-based platforms. The paper-based biosensor array and its electrochemical reader will enable the acquisition of high-density, statistically meaningful diagnostic information at the point of care in a rapid and cost-efficient way.     

Escherichia coli genosensor based on polyaniline

Avidin-modified polyaniline (PANI) electrochemically deposited onto a Pt disk electrode has been utilized for direct detection of Escherichia coli by immobilizing a 5'-biotin-labeled E. coli probe (BdE) using a differential pulse voltammetric technique in the presence of methylene blue as a DNA hybridization indicator. Depending on the target sample and the sonication time, this BdE-avidin-PANI bioelectrode can be utilized to electrochemically detect a complementary target probe (0.009 ng/microL), E. coli genomic DNA (0.01 ng/microL) and 11 E. coli cells/mL in 60 s to 14 min (hybridization time) without using PCR and can be used 5-7 times at temperatures of 30-45 degrees C.     

Microtubule sensors and sensor array based on polyaniline synthesized in the presence of poly(styrene sulfonate)

Microtubule sensors for glucose, urea, and triglyceride were fabricated based on poly(styrene sulfonate)-polyaniline (PSS-PANI) composites synthesized within the pores of track-etched polycarbonate membranes. The synthesis of a sufficiently thick and conducting PSS-PANI film at pH 5 provided the advantage of immobilizing enzymes during polymerization. This resulted in the improvement of sensor response for urea and triglyceride by a factor of approximately 10(2) with a significant increase in the linear region of response compared to polyaniline-based sensors, where the enzymes were immobilized by physical adsorption after the polymerization. The sensors based on urea and triglyceride were found to have a higher linear range of response, better sensitivity, improved multiple use capability, and faster response time compared to the potentiometric and amperometric sensors based on polyaniline. A microtubule sensor array for glucose, urea, and triglyceride based on PSS-PANI was fabricated by immobilization of three different sets of enzymes on three closely spaced devices and its response was found to be free from cross-interference when a sample containing a mixture of the above analytes was analyzed in a single measurement.     

Glucose biosensor based on the microcantilever

Diagnosis and management of diabetes require quantitative and selective detection of blood glucose levels. We report a technique for micromechanical detection of biologically relevant glucose concentrations by immobilization of glucose oxidase (GOx) onto a microcantilever surface. Microfabricated cantilevers have traditionally found utility in atomic force microscope imaging. During the past decade, however, microcantilevers have been increasingly used as transducers in chemical-sensing systems. This paper describes the combination of this technology with enzyme specificity to construct a highly selective glucose biosensor. The enzyme-functionalized microcantilever undergoes bending due to a change in surface stress induced by the reaction between glucose in solution and the GOx immobilized on the cantilever surface. Experiments were carried out under flow conditions. The common interferences for glucose detection in other detection schemes have been tested and have shown to have no effect on the measurement of blood glucose level by this technique.     

Demonstration of four immunoassay formats using the array biosensor

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.     

A cholesterol biosensor based on gold nanoparticles decorated functionalized graphene nanoplatelets

The fabrication of a cholesterol biosensor using gold nanoparticles decorated graphene nanoplatelets has been reported. Thermally exfoliated graphene nanoplatelets act as a suitable support for the deposition of Au nanoparticles. Cholesterol biosensor electrodes have been constructed with nafion solubilized functionalized graphene nanoplatelets (f-G) as well as Au nanoparticles decorated f-G, immobilized over glassy carbon electrode. f-G and Au/f-G thin film deposited glassy carbon electrodes were further functionalized with cholesterol oxidase by physical adsorption. Au nanoparticles dispersed over f-G demonstrate the ability to substantially raise the response current. The fabricated electrodes have been tested for their electrochemical performance at a potential of 0.2 V. The fabricated Au/f-G based cholesterol biosensor exhibits sensitivity of 314 nA/μM cm² for the detection of cholesterol with a linear response up to 135 μM. Furthermore, it has been observed that the biosensor exhibits a good anti-interference ability and favorable stability over a month's period.     

A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons

We have prepared a novel optical fiber evanescent wave DNA biosensor using a newly developed molecular beacon DNA probe. The molecular beacons (MB) are oligonucleotide probes that become fluorescent upon hybridization with target DNA/RNA molecules. Biotinylated MBs have been designed and immobilized on an optical fiber core surface via biotin-avidin or biotin-streptavidin interactions. The DNA sensor based on a MB does not need labeled analyte or intercalation reagents. It can be used to directly detect, in real-time, target DNA/RNA molecules without using competitive assays. The sensor is rapid, stable, highly selective, and reproducible. We have studied the hybridization kinetics of the immobilized MB by changing the ionic strength of the hybridization solution and target DNA concentration. Our result shows divalent cations play a more important role than monovalent cations in stabilizing the MB stem hybrids and in accelerating the hybridization reaction with target DNA/RNA molecules. The concentration detection limit of the MB evanescent wave biosensor is 1.1 nM. The MB DNA biosensor has been applied to the analysis of specific gamma-actin mRNA sequences amplified by polymerase chain reaction.     

Optical biosensor based on hollow integrated waveguides

The first absorbance biosensor based on pure silicon hollow integrated waveguides is presented in this work. With the use of horseradish peroxidase (HRP) as a model recognition element, an enzymatic sensor for the measurement of hydrogen peroxide was fabricated, numerically simulated, and experimentally characterized. Waveguides with widths ranging from 50 to 80 microm, having a depth of 50 microm and lengths up to 5 mm were easily fabricated by just one photolithographic step. These were further modified by covalent immobilization of HRP using silanization chemistry. Simulation studies of the proposed approach showed a sensor linear behavior up to 300 microM H2O2 and a sensitivity of 2.7 x 10(-3) AU/microM. Experimental results were in good agreement with the simulated ones. A linear behavior between 10 and 300 microM H2O2, a sensitivity of 3 x 10(-3) AU/microM, and a signal-to-noise ratio around 20 dB were attained. Also, kinetic studies of the activity of the immobilized enzyme on the silicon waveguide surface gave an apparent Michaelis-Menten constant of 0.44 mM. The simple technology proposed in this work enables the fabrication of cost-effective, easy-to-use, miniaturized biosensor generic platforms, these being envisioned as excellent candidates for the development of lab-on-a-chip systems.     

A classification tool for N-way array based on SIMCA methodology

In the literature there are only few papers concerned with classification methods for multi-way arrays. The most common procedure, by far, is to unfold the multi-way data array into an ordinary matrix and then to apply the traditional multivariate tools for classification. As opposed to unfolding the data several possibilities exist for building classification models more directly based on the multi-way structure of the data. As an example, multi-way partial least squares discriminant analysis has been used as a supervised classification method, another alternative that has been investigated is to perform classification using Fisher's LDA or SIMCA on the score matrix from e.g. a PARAFAC or a Tucker model. Despite a few attempts of applying such multi-way classification approaches, no-one has looked into how such models are best built and implemented.In this work, the SIMCA method is extended to three-way arrays. Included in this work is also actual code that will work on general multi-way arrays rather than just three-way arrays. In analogy with two-way SIMCA, a decomposition model is separately built for the multi-way data for each class, using multi-way decomposition method such as PARAFAC or Tucker3. In the choice of the best class dimensionality, i.e. number of latent factors, both the results of cross-validation but mainly the sensitivity/specificity values are evaluated. In order to estimate the class limits for each class model, orthogonal and score distances are considered, and different statistics are implemented and tested to set confidence limits for these two parameters. Classification performance using different definitions of class boundaries and classification rules, including the use of cross-validated residuals and scores is compared.The proposed N-SIMCA methodology and code, besides simulated data sets of varying dimensionality, has been tested on two case studies, concerning food authentication tasks for typical food products.     

A practical vacuum sensor based on a ZnO nanowire array

We report a practical vacuum pressure sensor based on a ZnO nanowire array (NWA). An oriented single-crystal ZnO NWA was synthesized by electrodeposition. The device consists of two ITO glass plates coated with a ZnO NWA. Scanning electron microscopy (SEM) and the x-ray diffraction (XRD) pattern show that the as-grown ZnO NWAs are single-crystal and roughly oriented with the ZnO(002) plane parallel to the substrate. Through measuring the pressure dependent resistance of the sensor at different gas species and temperatures, we discovered that the resistance increases monotonically with vacuum pressure. This demonstrates that a practical vacuum sensor could be fabricated since measurements were carried out with a normal multimeter, with no need for the high sensitivity and costly equipment as routinely required in nanotechnology for extremely weak signals. Measurement at elevated temperature (300?°C) showed that the vacuum sensor is much stabler and more sensitive to O(2) pressure. The principle of the device relates to the adsorbed oxygen species on the large surface area of a ZnO NWA to form a resistive depletion layer at the nanowire (NW) surface.     

A wireless, remote query glucose biosensor based on a pH-sensitive polymer

This paper describes a wireless, remote query glucose biosensor using a ribbonlike, mass-sensitive magnetoelastic sensor as the transducer. The glucose biosensor is fabricated by first coating the magnetoelastic sensor with a pH-sensitive polymer and upon it a layer of glucose oxidase (GOx). The pH-responsive polymer swells or shrinks, thereby changing mass, respectively, in response to increasing or decreasing pH values. The GOx-catalyzed oxidation of glucose produces gluconic acid, inducing the pH-responsive polymer to shrink, which in turn decreases the polymer mass. In response to a time-varying magnetic field, a magnetoelastic sensor mechanically vibrates at a characteristic resonance frequency, the value of which inversely depends on sensor mass loading. As the magnetoelastic films are magnetostrictive, the vibrations launch magnetic flux that can be remotely detected using a pickup coil. Hence, changes in the resonance frequency of a passive magnetoelastic transducer are detected on a remote query basis, without the use of physical connections to the sensors.The sensitivity of the glucose biosensors decreases with increasing ionic strength; at physiological salt concentrations, 0.6 mmol/L of glucose can be measured. At glucose concentrations of 1-10 mmol/L, the biosensor response is reversible and linear, with the detection limit of 0.6 mmol/L corresponding to an error in resonance frequency determination of 20 Hz. Since no physical connections between the sensor and the monitoring instrument are required, this sensor can potentially be applied to in vivo and in situ measurement of glucose concentrations.