A novel chemiluminescence paper microfluidic biosensor based on enzymatic reaction for uric acid determination

In this work, chemiluminescence (CL) method was combined with microfluidic paper-based analytical device (μPAD) to establish a novel CL μPAD biosensor for the first time. This novel CL μPAD biosensor was based on enzyme reaction which produced H(2)O(2) while decomposing the substrate and the CL reaction between rhodanine derivative and generated H(2)O(2) in acid medium. Microchannels in μPAD were fabricated by cutting method. And the possible CL assay principle of this CL μPAD biosensor was explained. Rhodanine derivative system was used to reach the purpose of high sensitivity and well-defined signal for this CL μPAD biosensor. And the optimum reaction conditions were investigated. The quantitative determination of uric acid could be achieved by this CL μPAD biosensor with accurate and satisfactory result. And this biosensor could provide good reproducible results upon storage at 4°C for at least 10 weeks. The successful integration of μPAD and CL reaction made the final biosensor inexpensive, easy-to-use, low-volume, and portable for uric acid determination, which also greatly reduces the cost and increases the efficiency required for an analysis. We believe this simple, practical CL μPAD biosensor will be of interest for use in areas such as disease diagnosis.     

A new photoelectrochemical biosensors based on DNA conformational changes and isothermal circular strand-displacement polymerization reaction

We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to electrochemical DNA (E-DNA) approach. To demonstrate the effect of distance change for photosensitizer to the surface of electrode on the change of photocurrent, photosensitizer Ru(bpy)(2)(dcbpy)(2+) tagged DNA stem-loop structures were self-assembled onto a nanogold modified ITO electrode. Hybridization induced a large conformational change in DNA structure, which in turn significantly altered the electron-transfer tunneling distance between the electrode and photosensitizer. The resulting change in photocurrent was proportional to the concentration of DNA in the range of 1.0×10(-10)-8.0×10(-9)M. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With multiple rounds of isothermal strand replication, which led to strand displacement and constituted consecutive signal amplification, a detection limit of 9.4×10(-14)M target DNA was achieved.     

Electrogenerated chemiluminescence detection of adenosine based on triplex DNA biosensor

A novel electrogenerated chemiluminescence (ECL) biosensor based on the construction of triplex DNA for the detection of adenosine was designed. The ECL biosensor employs an aptamer as a molecular recognition element, and quenches ECL of tris(2,2'-bipyridine) ruthenium (Ru(bpy)(3)(2+)) by ferrocenemonocarboxylic acid (FcA). Through self-assembly technology, the ECL probe of thiolated hairpin adenosine aptamer tagged was self-assembled onto the surface of a gold electrode with an ECL signal producer Ru(bpy)(3)(2+) derivative (Ru-DNA-1). The adenosine aptamer, including a section of triplex characteristic chain, formatted triplex DNA with two other DNAs (DNA-2, Fc-DNA-3) in the presence of triplex DNA binder coralyne chloride (CORA). Fc-DNA-3 was tagged with an ECL quencher ferrocenemonocarboxylic acid (FcA), a quenching probe. In the presence of adenosine, the aptamer sequence (Ru-DNA-1) prefers to form the aptamer-adenosine complex with hairpin configuration and the switch of the DNA-1 occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of a quenching probe. Meanwhile, a control experiment was performed; the ECL-duplex biosensor was designed to detect adenosine. The detection limits were 2.7×10(-10) mol L(-1) and 2.3×10(-9) mol L(-1) for the ECL-triplex DNA biosensor and ECL-duplex DNA biosensor, respectively, which demonstrated that the ECL-triplex DNA biosensor improved the sensitivity and specificity greatly.     

New luminol chemiluminescence reaction using diperiodatoargentate as oxidate for the determination of amikacin sulfate

A new chemiluminescence (CL) reaction between luminol and diperiodatoargentate {K₂ [Ag (H₂IO₆) (OH) ₂]} was observed in alkaline medium. The CL intensity could be greatly enhanced by amikacin sulfate. Therefore a new CL method for the determination of amikacin sulfate was built by combining with flow injection technology. A possible mechanism of the CL reaction was proposed via the investigation of the CL kinetic characteristics, the CL spectrum and the UV absorption spectra of some related substance. The concentration range of linear response was 5.1 × 10^?8 to 5.1 × 10^?6 mol L^?1 with a detection limit of 1.9 × 10^?8 mol L^?1 (3σ). The proposed method had good reproducibility with a relative standard deviation of 2.8% (n = 7) for 5.1 × 10^?7 mol L^?1 of amikacin sulfate. It was successfully applied to determine amikacin sulfate in serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor

A new biosensor employing immobilized DNA on a nano-structured conductive polymer fixed onto a platinum electrode is presented. Upon optimization of synthesis parameters, polypyrrole nanofibers, 30-90 nm in diameter, were synthesized in an aqueous media by the electropolymerization of pyrrole using normal pulse voltammetry (NPV). The nanofiber film was investigated by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Double-stranded DNA was physisorbed onto the PPy nanofiber films. Various parameters, including the pH and DNA concentration, were optimized. The DNA immobilized on the nanofiber films was characterized using differential pulse voltammetry (DPV) and Fourier-transform infrared (FTIR) spectroscopy. Using DPV to study the interaction of spermidine with DNA, a binding constant (K) value of 4.08 x 10(5)+/-0.05 M(-1) was obtained. For the determination of spermidine, the proposed method exhibited a good dynamic range, correlation coefficient (0.05-1.0 microM and 0.9983, respectively) and a low detection limit (0.02 microM), although Ca(2+) ions were found to electrostatically bind to DNA and weaken the spermidine-DNA interaction.     

Enhanced photoelectrochemical method for linear DNA hybridization detection using Au-nanopaticle labeled DNA as probe onto titanium dioxide electrode

A photoelectrochemical method was proposed to detect DNA hybridization using Au nanoparticle modified DNA as one probe on TiO2 substrate, in which the TiO2 substrate was used not only as DNA anchors but also as the signal transducers. Hybridization between the probe and the target DNA oligonucleotides was confirmed by the decreased photocurrent of the TiO2 electrode. Compared with non-label probe, Au nanoparticles enhanced the photocurrent shifts after the hybridization. The photocurrent decreased with increasing the concentration of target DNA, indicating that this method could be used for quantitative measurements, and the discrimination of the complementary from mismatched DNA. Furthermore, the hybridization binding constant was obtained and photocurrent generation mechanism was discussed. The major advantages of this photochemical method are speed, simplicity and excellent specificity. This method provides a platform for studying a wide variety of biological processes using photoelectrochemical method.     

A fiber-optic microarray biosensor using aptamers as receptors

A fiber-optic biosensor using an aptamer receptor has been developed for the measurement of thrombin. An antithrombin DNA aptamer was immobilized on the surface of silica microspheres, and these aptamer beads were distributed in microwells on the distal tip of an imaging fiber. A different oligonucleotide bead type prepared using the same method as the aptamer beads was also included in the microwells to measure the degree of nonspecific binding. The imaging fiber was coupled to a modified epifluorescence microscope system, and the distal end of the fiber was incubated with a fluorescein-labeled thrombin (F-thrombin) solution. Nonlabeled thrombin could be detected using a competitive binding assay with F-thrombin. The aptamer beads selectively bound to the target and could be reused without any sensitivity change. The fiber-optic microarray system has a detection limit of 1 nM for nonlabeled thrombin, and each test can be performed in ca. 15 min including the regeneration time.     

A new biosensor for osteoporosis detection

Abstract

Osteoporosis is a disease that is characterized by deterioration of bone tissue and increased risk of fracture as it leads to a decrease in bone mineral density, which is an important public health problem. Today, bone mineral density is measured by radiological techniques. Alternative techniques are needed because of the disadvantages such as excessive radiation intake, the cost of radiological techniques, and the necessity for specialist personnel for the devices. The quantitative determination of biochemical markers that play a role in bone mineralization may be a good alternative for the osteoporosis diagnosis and especially in the follow-up of treatment.

In this study, a specific and sensitive immunological biosensor, which quantitatively determines the osteocalcin molecule, has been developed to be used in the early osteoporosis diagnosis and to evaluate the response to the drug treatment. Anti-osteocalcin antibody was immobilized onto gold electrode surface via covalent immobilization method by using 6-mercaptohexanol, 1,4-butanedioldiglycidyl ether, ethanolamine, and glutaraldehyde. Immobilization steps and biosensor characterization were specified by cyclic voltammetry and electrochemical impedance spectroscopy. The detection time and range of Ocn biosensor were determined as 45?min and 10–60?pg µL^?1 Ocn concentration, respectively. The Ocn biosensor was successfully applied in artificial serum samples spiked with Ocn.     

Electrogenerated chemiluminescence biosensor incorporating ruthenium complex-labelled Concanavalin A as a probe for the detection of Escherichia coli

The interaction of riboflavin with salmon sperm double-stranded DNA based on the decreasing of the oxidation signal of guanine and adenine bases was studied electrochemically with a pencil graphite electrode (PGE) using differential pulse voltammetry. The decrease in the intensity of the guanine and adenine oxidation signals after interaction with riboflavin was used as an indicator signals for the sensitive determination of riboflavin. Under the optimum conditions, a linear dependence of the guanine and adenine oxidation signals was observed for the riboflavin concentration in the range of 0.5-70 μg mL(-1) with a detection limit of 0.34 μg mL(-1) at ds-DNA modified PGE. The reproducibility and applicability of the analysis to pharmaceutical dosage forms and urine sample were also investigated. These results showed that this DNA biosensor could be used for the sensitive, rapid, simple and cost effective detection and determination of riboflavin-ds-DNA interaction. Pretreated pencil graphite electrode (PPGE) was also used for the determination of riboflavin by differential pulse adsorptive stripping voltammetry. With PPGE, a linear relationship was obtained for riboflavin over the concentration range of 0.003-0.88 μg mL(-1) with differential pulse adsorptive stripping voltammetric signal and with a detection limit of 0.076 ng mL(-1). Both determination methods were fully validated and applied for the analysis of riboflavin.     

Hairpin DNA probe based electrochemical biosensor using methylene blue as hybridization indicator

In this paper, a label-free, rapid and simple method was proposed to study the hybridization specificity of hairpin DNA probe using methylene blue (MB) as a hybridization indicator. Thiolated hairpin DNA probe was immobilized on the gold electrode by self-assembly. The voltammetric signals of MB were investigated at these modified electrodes by means of cyclic voltammetry (CV) detection. Single-base mutation oligonucleotide and random oligonucleotide can be easily discriminated from complementary target DNA. The effect of mismatch position in target DNA was investigated. Experimental results showed that mutation in the center of target DNA had greatest effect on the hybridization with hairpin DNA probe. The relationship between electrochemical responses and DNA target concentration was also studied. The reduction current of MB intercalation decreased with increasing the concentration of target DNA. Taken together, these experiments demonstrate that the hybridization indicator MB provides great promise for rapid and specific measurement of target DNA.     

Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.     

Monitoring glutamine in animal cell cultures using a chemiluminescence fiber optic biosensor

Together with flow injection analysis (FIA), a chemiluminescence (CL) fiber optic biosensor system has been developed for determining glutamine in animal cell cultures. Glutaminase (GAH) and glutamate oxidase (GLO) were onto separate porous aminopropyl glass beads via glutaraldehyde activation and packed to form an enzyme column. These two enzymes acted in sequence on glutamine to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. An anion exchanger was introduced on-line to eliminate interfering endogenous glutamate in view of its negative charge at pH above 3.22 (isoelectric pH). Among several resins tested, the acetate form was most effective, and this type of ion exchanger also effectively adsorbed uric acid, acetaminophen, and aspartic acid.There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 1 to 100 muM. A complete analysis could be performed in 2 min, including sampling and washing with a good reproducibility (+/- 4.4%). Both the bi-enzymic and ion exchange columns were useful for at least 500 analyses when the biosensor system was applied for the glutamine determination in murine hybridoma cell cultures and insect cell cultures. The values obtained compared well with those of HPLC, thus validating the applicability of the CL fiber optic system. (c) 1993 John Wiley & Sons, Inc.     

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its array for the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-1-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA ofp53 gene,N-ras gene andRb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.     

Development of an oxygen-rich biosensor using enzymatic reaction

This work reports a novel strategy for the development of an O₂-rich biosensor. The principle is based on an enzymatic reaction between catalase and H₂O₂ to release O₂, thus to increase the O₂ amount in the enzyme matrix. This method improves the determination reliability by alleviating the O₂ dependence.     

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its array for the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-l-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA of p53 gene, N-ras gene and Rb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.     

A new interference-free lysine biosensor using a non-conducting polymer film

An electrochemical biosensor for the determination of lysine to be used for rapid evaluation of food quality has been developed. Platinum electrodes have been coated by electropolymerisation with 1,2-diaminobenzene (1.2-DAB) using cyclic voltammetry. The reduction in the oxidation of interferents compared with the bare platinum electrode was 100% for ascorbic acid, 99% for acetaminophen and 99% for cysteine. The enzyme L-lysine--oxidase was then immobilised onto the polymer layer by passive adsorption and a calibration curve for lysine constructed. This gave a linear range of 1×10⁻⁵ mol/l to 1×10⁻³ mol/l and a limit of detection of 2×10⁻⁷ mol/l.