A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.

     

Biotechnological storage and utilization of entrapped solar energy

Our laboratory has recently developed a device employing immobilized F₀F₁ adenosine triphosphatase (ATPase) that allows synthesis of adenosine triphosphate (ATP) from adenosine 5′-diphosphate and inorganic phosphate using solar energy. We present estimates of total solar energy received by Earth’s land area and demonstrate that its efficient capture may allow conversion of solar energy and storage into bonds of biochemicals using devices harboring either immobilized ATPase or NADH dehydrogenase. Capture and storage of solar energy into biochemicals may also enable fixation of CO₂ emanating from polluting units. The cofactors ATP and NADH synthesized using solar energy could be used for regeneration of acceptor d-ribulose-1,5-bisphosphate from 3-phosphoglycerate formed during CO₂ fixation.     

Determination of radon using solid state nuclear tracks wireless sensing method

A highly sensitive and selective electrochemiluminescent (ECL) biosensor for the determination of adenosine was developed. Single DNA (capture DNA) was immobilized on the gold electrode through Au-thiol interaction at first. Another DNA modified with tris(2,2'-bipyridyl) ruthenium(II)-doped silica nanoparticles (Ru-SNPs) that contained adenosine aptamer was then modified on the electrode surface through hybridizing with the capture DNA. In the presence of adenosine, adenosine-aptamer complex is produced rather than aptamer-DNA duplex, resulting with the dissociation of Ru-SNPs-labeled aptamer from the electrode surface and the decrease in the ECL intensity. The decrease of ECL intensity has a direct relationship with the logarithm of adenosine concentration in the range of 1.0×10(-10) to 5.0×10(-6)molL(-1). The detection limit of the proposed method is 3.0×10(-11)molL(-1). The existence of guanosine, cytidine and uridine has little interference with adenosine detection, demonstrating that the developed biosensor owns a high selectivity to adenosine. In addition, the developed biosensor also demonstrates very good reusability, as after being reused for 30 times, its ECL signal still keeps 91% of its original state.     

Adenosine Reagentless Electrochemical Aptasensor Using a Phosphorothioate Immobilization Strategy

This work deals with the characterization of a phosphorothioate anchoring strategy for aptamer molecules linked to gold, in the context of electrochemical sensors, using adenosine aptamer as model system. Surface density of immobilized phosphorothioate oligonucleotide sequences has been explored for a range of oligonucleotide concentrations (0.055–55 μM), finding a consequent variation of molecular surface density (3.5×10¹¹–2.8×10¹³ molecules/cm²). Most suitable aptamer concentration for adenosine recognition was also explored and found to be around 5.5 μM. As proof of concept of phosphorothioate strategy, electrochemical response to adenosine concentration was measured using a ferrocene‐labeled oligonucleotide sequence, and phosphorothioate anchoring thermal stability was compared to thiol immobilization.     

Au NPs-aptamer conjugates as a powerful competitive reagent for ultrasensitive detection of small molecules by surface plasmon resonance spectroscopy

Features of Au NPs-aptamer conjugates as a powerful competitive reagent to substitute antibody in enhancing surface plasmon resonance spectroscopy (SPR) signal for the detection of small molecule are explored for the first time. In order to evaluate the sensing ability of Au NPs-aptamer conjugates as a competitive reagent, a novel SPR sensor based on indirect competitive inhibition assay (ICIA) for the detection of adenosine is constructed by employing the competitive reaction between antiadenosine aptamer with adenosine and antiadenosine aptamer with its partial complementary ss-DNA. The partial complementary ss-DNA of antiadenosine aptamer is firstly immobilized on SPR gold film as sensing surface. When the Au NPs-antiadenosine aptamer conjugates solution is added to SPR cell in the absence of adenosine, Au NPs-antiadenosine aptamer conjugates is adsorbed to SPR sensor by the DNA hybridization reaction, and results in a large change of SPR signal. However, the change of SPR signal is decreased when the mixing solution of adenosine with Au NPs-antiadenosine aptamer conjugates is added. This is because adenosine reacts with antiadenosine aptamer in Au NPs-antiadenosine aptamer conjugates and changes its structure from ss-DNA to tertiary structure, which cannot hybridize with its partial complementary ss-DNA immobilized on SPR gold surface. Based on this principle, a SPR sensor for indirect detection of adenosine can be developed. The experimental results confirm that the SPR sensor possesses a good sensitivity and a high selectivity for adenosine, which indirectly confirms that Au NPs-aptamer conjugates is a powerful competitive reagent. More significantly, it can be used to develop other SPR sensors based on ICIA to detect different targets by changing the corresponding type of aptamer in Au NPs-aptamer conjugates.     

Rapid assays based on immobilized bioluminescent enzymes and photographic detection of light-emission

Sensitive assays have been developed for adenosine 5'-triphosphate, the reduced form of nicotinamide adenine dinucleotide, cholyl glycine and alcohol, with immobilized and co-immobilized preparations of bacterial and firefly luciferase as reagents. With high-speed (ASA 20000) instant photographic film as detector, picomole amounts of the various analytes can be detected rapidly. The simplicity and convenience of the analytical combination of co-immobilized bioluminescent enzymes and photographic film for the detection of light make this an ideal technique for rapid screening tests.     

Affinity chromatography in purification of A1 adenosine receptors.

Purification of A1 adenosine receptor of rat brain membranes was performed using a newly developed affinity gel employing xanthine amine congener (XAC) as an immobilized ligand. The A1 adenosine receptor was solubilized with digitonin-cholate from brain membranes and then purified by a sequential use of affinity chromatography on XAC-agarose, hydroxyapatite chromatography and reaffinity chromatography on XAC-agarose. The A1 adenosine receptor was purified ca. 45,000-fold with a yield of 5%. The final receptor preparation gave a single broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr approximately 34,000. This band was also shown to be specifically labelled with an affinity labelling reagent for A1 adenosine receptors. This purification method was also applicable for the complete purification of A1 adenosine receptors from rat testis and human brain membranes.     

Flow-injection determination of adenosine and inosine in blood plasma with immobilized enzyme columns connected in series and fluorimetric detection

A rapid and sensitive flow-injection method is described for the enzymatic determination of adenosine and inosine in human blood plama. Teflon columns prepared by packing adenosine deaminase. purine nucleoside phosphorylase, xanthine oxidase, uricase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of adenosine and inosine is measured fluorimetrically after reaction with 3-(p-hydroxyphenyl)propionic acid. Linear calibrations were obtained for 0.5–500 pmol of adenosine or inosine in hte 20 μl sample injected. Necessary deproteination routines are outlined.     

Sensitive chemiluminescence aptasensor based on exonuclease-assisted recycling amplification

We report herein an exonuclease-assisted aptamer-based target recycling amplification strategy for sensitive and selective chemiluminescence (CL) determination of adenosine. This aptasensor is based on target-induced release of aptamers from capture probes immobilized on the 96-well plate surface, and thus leading to a decreased hybridization with gold nanoparticle-functionalized reporter sequences followed by a CL signal. The introduction of exonuclease III catalyzes the stepwise removal of mononucleotides from 3′-hydroxyl termini of duplex DNAs of aptamers, liberating the adenosine. Therefore, a single copy of target adenosine can lead to the release and digestion of numerous aptamer strands from the 96-well plates and ultimately an enhanced sensitivity is achieved. Experimental results revealed that the exonuclease-assisted recycling strategy enabled the monitoring of adenosine with wide working ranges and low detection limits (LOD: 0.5 nM). This new CL strategy might create a novel technology for the detection of various targets and could find wide applications in the environmental and biomedical fields.     

Functionalized electrospun mats from styrene-maleic anhydride copolymers for immobilization of acetylcholinesterase

A series of electrospun mats of styrene-maleic anhydride copolymers has been functionalized by modification with two types of spacers - a polymer with a flexible hydrophilic polyether chain (Jeffamine® ED) and a rigid low-molecular-weight spacer (р-phenylenediamine). The successful modification of the microfibrous mats with these bifunctional compounds has been proved by ATR-FTIR and X-ray photoelectron spectroscopy (XPS) analyses. In a further step, acetylcholinesterase (AChE) has been covalently immobilized onto the modified mats using glutaraldehyde as a binding agent. The amount of bound protein and the specific activity of the immobilized AChE have been determined. The highest activity has been displayed by AChE covalently bound to Jeffamine-modified microfibrous mats. Moreover, the immobilized AChE is characterized by enhanced thermal and storage stability as compared to the free enzyme.     

Aptamer-based Electrochemical Biosensors for Highly Selective and Quantitative Detection of Adenosine

A new adenosine biosensor based on aptamer probe is introduced in this article. An amino-labeled aptamer probe was immobilized on the gold electrode modified with an o-phenylenediamine electropolymerized film. When adenosine is bound specifically to the aptamer probe, the interface of the biosensor is changed, resulting in the decrement of the peak current. The response current is proportional to the amount of adenosine in sample. The used electrode can be easily regenerated in hot water. The proposed biosensor represents a linear response to adenosine over a concentration range of 1.0x 10^-7-l.0x10^-4 mol/L with a detection limit of 1.0xl0^-8 mol/L. The presented biosensor exhibits a nice specificity towards adenosine. It offers a promising approach for adenosine assay due to its excellent electrochemical properties that are believed to be very attractive for electrochemical studies and electroanalytical applications.     

A simple non-enzymatic strategy for adenosine triphosphate electrochemical aptasensor using silver nanoparticle-decorated graphene oxide

In this work, a sensitive electrochemical aptasensor for the detection of adenosine triphosphate (ATP) has been introduced. A simple and non-enzymatic signal amplification strategy is utilized using silver nanoparticle-decorated graphene oxide (AgNPs–GO) as a redox probe. The modified electrode surface was characterized by scanning electron microscopy, FTIR and UV–Vis spectroscopy, and electrochemical impedance spectroscopy. GO provides an excellent substrate for the presence of the large number of AgNPs, so the monitored oxidation signal of AgNPs has been amplified. ATP-specific DNA aptamer is split into two fragments (F₁ & F₂) in order to design a sandwich-type assay. For the construction of the sensor, the surface of a graphite screen-printed electrode is modified with electrodeposited gold nanoparticles followed by self-assembling a monolayer of 3-mercaptopropionic acid on the electrode surface. The first amino-labeled fragment, F₁, is immobilized on the modified electrode via carbodiimide chemistry. The synthesized AgNPs–GO interacts with F₁ via \(\pi{-}\pi\) stacking. In the presence of ATP, the second fragment of the aptamer, F₂, forms an associated complex with the immobilized F₁ and causes AgNPs–GO to leave the surface. Consequently, a remarkable decrease in the oxidation signal of the AgNPs is observed. The percentage of this decrease has been monitored as an analytical signal, which is proportional to ATP concentration, and delivers a linear response over the range of 10.0 (±0.6) to 850 (±5) nM with a detection limit of 5.0 (±0.2) nM.     

固定化谷氨酸脱羧酶性能的研究

本文首镒用羧甲基化的以Ｎ,Ｎ’－甲叉双丙烯酰胺交联的烯丙基葡聚糖凝胶生化树脂为新型载体,将谷氨酸脱羧酶固定在ＣＭ－ＣＡＤＢ树脂上。研究了固定化ＧＤＣ的活性与低物浓度,ｐＨ,温度的依存性;动态响应特性;热稳定性和寿命;求算了米氏常数和反应活化能;将固定化ＧＤＣ酶柱与进样系统－离子活度分析器－计算机数据采集系统匹配,构成酶传感器谷氨酸检测装置,测定了固定化ＧＤＣ酶柱的能斯特线性响应曲线,线性方程为ｙ／     

Glucose oxidase—hexokinase bienzyme electrode sensor for adenosine triphosphate

A bienzyme electrode, based on the oxygen electrode, and containing immobilized glucose oxidase and hexokinase is described for the measurement of 0.2–3 mM adenosine triphosphate.     

A Dual-Enzyme Electrode for the Determination of Flavin Adenine Dinucleotide in the Presence of Riboflavin and Flavin Mononucleotide

Abstract

An electrode method has been developed for the determination of flavin adenine dinucleotide (FAD) using a potentiometric gas sensor and commercially available enzyme preparations. The construction of the FAD-sensitive electrode is based on immobilizing alkaline phosphatase (E.C. 3.1.3.1) and adenosine deaminase (E.C. 3.5.4.4) on the sensing tip of an ammonia gas sensor. Alkaline phosphatase enzymatically catalyzes the hydrolysis of FAD to adenosine which is subsequently converted to ammonia by adenosine deaminase. The response of the dual-enzyme electrode is linear between 8 × 10^?5 M and 4 × 10^?3 M FAD with a slope of 43 mV/decade concentration at pH 8.5 and 37[ddot]C. The optimum buffer system is 0.5 M diethanolamine, 1 × 10^?3 M Tris-HCl and 1 × 10^?3 M MgCl₂. Electrodes constructed with enzymes immobilized by cross-linking with glutaraldehyde and bovine serum albumin showed longer life times than electrodes using enzymes entrapped by a dialysis membrane. The electrode is highly selective over riboflavin and flavin mononucleotide, but it does respond to other nucleotides.     

Cobalt(II) complexation with immobilized proteinases of Candida albicans

Complexation of cobalt(II) ion with proteolytic enzymes of Candida albicans (SAP C.alb.) of induced (ISAP C.alb.) and constitutive (CSAP C.alb.) types immobilized on the cellulose nitrate membrane surface has been studied. The maximal sorption capacity of cellulose nitrate membranes with covalently immobilized induced proteinase ISAP C.alb. with respect to Co(II) ions is 16.5 ??mol/cm², and that for CSAP C.alb. is 27.7 ??mol/cm². The model of fixed polydentate centers has been used for describing complex formation. The complexation constants (?? _n ) and the average number of immobilized ligands coordinated to one metal atom (n) have been determined. The specificity of binding of immobilized enzyme molecules to Co(II) ion has been assessed.     

GPCRs as therapeutic targets: a view on adenosine receptors structure and functions, and molecular modeling support

G-protein-coupled receptors (GPCRs) represent the largest known family of signal-transducing proteins and transmit signals for light and many extracellular regulatory molecules. GPCRs are dysfunctional or dysregulated in several human diseases and are estimated to be the targets of ～40% of the drugs used in clinical medicine today. Receptors for adenosine belong to this family, and so far four subtypes, the A₁, A_2A, A_2B, and A₃, have been recognized. The activation of adenosine receptors (ARs) is largely responsible for the variety of effects produced by adenosine throughout several organ systems. Based on the wide (and often beneficial) effects attributed to the accumulation of endogenously released adenosine, it has long been considered that regulation of ARs has considerable therapeutic potential. In this review, we focus on recent work on adenosine receptors as therapeutic targets and, in particular, on molecular modelling support to adenosine receptors targeting.     

Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel

A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 microg ml(-1)) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 microm width x 6 microm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 microM) from being transported to the electrode surface while nitric oxide (NO, 10 microM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 microM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.     

Speciation of chromium by selective separation and preconcentration of Cr(III) on an immobilized nanometer titanium dioxide microcolumn

Nanometer titanium dioxide immobilized on silica gel (immobilized nanometer-scale TiO2 particles) was prepared by a sol-gel method and characterized by X-ray diffraction and scanning electron microscopy. The adsorptive behavior of Cr(III) and Cr(VI) on immobilized nanometer TiO2 was assessed. Cr(III) was selectively sorbed on immobilized nanometer TiO2 in the pH range of 7-9, while Cr(VI) was found to remain in solution. A sensitive and selective method has been developed for the speciation of chromium in water samples using an immobilized nanometer TiO2 microcolumn and inductively coupled plasma atomic emission spectrometry. Under optimized conditions (pH 7.0, flow rate 2.0 mL/min), Cr(III) was retained on the column, then eluted with 0.5 mol/L HNO3 and determined by ICP-AES. Total chromium was determined after the reduction of Cr(VI) to Cr(III) by ascorbic acid. The adsorption capacity of immobilized nanometer TiO2 for Cr(III) was found to be 7.04 mg/g. The detection limit for Cr(III) was 0.22 ng/mL and the RSD was 3.5% (n = 11, c = 100 ng/ mL) with an enrichment factor of 50. The proposed method has been applied to the speciation of chromium in water samples with satisfactory results.     

Fluorescence Detection of Adenosine Triphosphate in an Aqueous Solution Using a Combination of Copper(II) Complexes

Fluorescent ligands have been designed to form ternary complexes with a Cu(II) cation and phosphates in a buffer solution at physiological pH 7.4. It has been shown that a combination of two different ligands and CuCl(2) allows one to achieve high adenosine triphosphate/adenosine diphosphate, adenosine 5'-monophosphate selectivity, and ratiometric fluorescence sensing, while separately each ligand complex does not have such properties.