Mid-infrared spectroscopic measurement of ionic dissociative materials in the metabolic pathway

We determine the pH dependency of the mid-infrared spectra in aqueous solution of the organic dissociative materials in the metabolic pathway: saccharide phosphates (G6P, F6P), adenosine, and its phosphates (ATP, ADP, AMP). The series of molar absorbance spectra for these reagents were obtained in a pH range of about 2 to 11 with a Fourier transform infrared (FT-IR) spectrometer equipped with a horizontal diamond attenuated total reflection (ATR) sampling accessory. We also provide a method of infrared spectral extraction of ionic dissociative materials by performing a linear least-square fitting utilizing the formulas of ionic dissociation equilibrium shift, and we obtain the infrared spectrum of each ionic species of the dissociative materials: G6P-, G6P2-; F6P-, F6P2-; ATP2-, ATP3-, ATP4-; ADP-, ADP2-, ADP3-; AMP, AMP-, AMP2-; and adenosine+, adenosine0. The infrared spectral structure of each ionic species of the dissociative materials in the metabolic pathway are discussed. Additionally, the possibility for a quantification system of the concentrations of the organic dissociative materials in varying pH is suggested.     

Subsecond detection of physiological adenosine concentrations using fast-scan cyclic voltammetry

Adenosine modulates blood flow and neurotransmission and may be protective during pathological conditions such as ischemia and stroke. A real-time sensor of adenosine concentrations is needed to understand its physiological actions and the extent of receptor activation. Microelectrodes are advantageous for in vivo measurements because they are small and can make fast measurements. The goal of this study was to characterize detection of physiological adenosine concentration changes at carbon-fiber microelectrodes with subsecond temporal resolution. The oxidation potential of adenosine is +1.3 V, so fast-scan cyclic voltammetry (FSCV) was performed with an applied potential from -0.4 to 1.5 V and back at 400 V/s every 100 ms. Two oxidation peaks were detected for adenosine with T-650 carbon fibers. The second oxidation peak at 1.0 V occurs after the initial oxidation at 1.5 V and is due to a sequential oxidation step. Adsorption was maximized to obtain detection limits of 15 nM, lower than basal adenosine concentrations in the brain. The electrode was insensitive to the metabolite inosine and seven times more sensitive to adenosine than ATP. The enzymatic degradation of adenosine was monitored with FSCV. This microelectrode sensor will be valuable for biological monitoring of adenosine.     

Fluorescence determination of nitric oxide production in stimulated and activated platelets

Nitric oxide (NO) is quantitatively determined in platelets prior to, and after, stimulation with adenosine triphosphate (ATP) or activation with adenosine diphosphate (ADP). Platelets obtained from the whole blood of rabbits were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent NO production was measured as a fluorescent benzotriazole. Experiments were performed to determine the effect of probe concentration and probe incubation time in the platelets prior to measurement of the fluorescence. This information, combined with the method of multiple standard additions, was then employed to determine the moles of intracellular NO in the platelets (2.7 +/- 0.3) x 10(-16) mol of NO/platelet and the basal level of extracellular NO in the platelet sample (9.9 +/- 2.2) x 10(-18) mol of NO/platelet. Moreover, this method was used to quantitatively determine the amount of NO released from platelets whose NO production was stimulated with ATP (a nitric oxide synthase stimulus) or ADP, a substance known to result in NO production through platelet aggregation. When stimulated with ATP, the NO released from the platelets was determined to be (2.0 +/- 0.1) x 10(-17) mol of NO/platelet. When activated with ADP, the platelets released (2.8 +/- 0.3) x 10(-17) mol of NO/platelet. The difference between the extracellular basal levels of NO and that after stimulation with either ATP or ADP is in agreement with current estimates of NO release from platelets. Therefore, we conclude that a fluorescence determination of NO using the DAF family of probes, in combination with the method of multiple standard additions, can be employed to quantitatively determine the basal levels of NO in platelets, as well as the amount of NO released from stimulated and/or activated platelets.     

Enzyme colorimetric assay using unmodified silver nanoparticles

Colorimetric assay based on the unique surface plasmon resonance properties of metallic nanoparticles has received considerable attention in bioassay due to its simplicity, high sensitivity, and low cost. Most of colorimetric methods previously reported employed gold nanoparticles (GNPs) as sensing elements. In this work, we develop a sensitive, selective, simple, and label-free colorimetric assay using unmodified silver nanoparticle (AgNP) probes to detect enzymatic reactions. Enzymatic reactions concerning adenosine triphosphate (ATP) dephosphorylation by calf intestine alkaline phosphatase (CIAP) and peptide phosphorylation by protein kinase A (PKA) were studied. In the absence of the enzymes, unreacted ATP could protect AgNPs from salt-induced aggregation, whereas in the presence of the enzymes, the reaction product of ATP (i.e., adenosine for CIAP and ADP for PKA) could not. Via our method, dephosphorylation and phosphorylation could be readily detected by the color change of AgNPs, with a detection limit of 1 unit/mL for CIAP and a detection limit of 0.022 unit/mL for PKA. More importantly, the enzymatic inhibition by inhibitors and enzymatic activity in complex biological fluids could also be realized. This work is an important step toward a colorimetric assay using AgNPs and might provide a promise for enzyme assay in realistically complex systems and for screening of different enzyme inhibitors in future.     

Luciferase-based assay for adenosine: application to S-adenosyl-L-homocysteine hydrolase

S-Adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosyl-L-homocysteine (SAH) to adenosine (ADO) and L-homocysteine, promoting methyltransferase activity by relief of SAH inhibition. SAH catabolism is linked to S-adenosylmethionine metabolism, and the development of SAHH inhibitors is of interest for new therapeutics with anticancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96-well-plate format. The high-affinity adenosine kinase from Anopheles gambiae efficiently converts adenosine to adenosine monophosphate (AMP) in the presence of guanosine triphosphate. AMP is converted to adenosine triphosphate and coupled to firefly luciferase. With this procedure, kinetic parameters (K(m), k(cat)) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultrasensitive detection (10(-7) unit of SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions.     

Retention and separation of adenosine and analogues by affinity chromatography with an aptamer stationary phase

A biotinylated-DNA aptamer (molecular weight 16,600) that binds adenosine and related compounds in solution was immobilized by reaction with streptavidin, which had been covalently attached to porous chromatographic supports. The aptamer medium was packed into fused-silica capillaries (50-150-microm i.d.) to form affinity chromatography columns. Frontal chromatography analysis indicated that the dissociation constants (Kd) of cyclic-AMP, AMP, ATP, ADP, and adenosine were 138 +/- 18, 58 +/- 2, 38 +/- 2, 28 +/- 6 and 3 +/- 1 microM, respectively, for aptamer immobilized on a controlled pore glass support. Similar values were obtained for aptamer immobilized on a polystyrene support except for a slightly higher Kd for adenosine. The Kd for adenosine is similar to the previously reported value of 6 +/- 3 microM for adenosine-aptamer in solution indicating that immobilized aptamers can have affinity similar to that of the solution forms. Columns had 20 nmol of binding sites/100 microL of support media, which is 3.3-fold higher than that previously reported for immobilization of IgG on similar media, indicating that the aptamer can be immobilized with higher density than antibodies. Variation of mobile-phase conditions revealed that ionic strength and Mg2+ level had strong effects on retention of analytes while pH and buffer composition had less of an effect. It was demonstrated that the column could selectively retain and separate cyclic-AMP, NAD+, AMP, ADP, ATP, and adenosine, even in complex mixtures such as tissue extracts.     

Ruthenium purple-mediated microelectrode biosensors based on sol-gel film

Ruthenium Purple (RP), an analogue of Prussian Blue, has potentially advantageous electrochemical characteristics. We now demonstrate its use in microelectrode biosensors for the first time. An RP layer was grown on, and remained stably anchored to, the surface of gold microelectrodes at physiological pH ranges. Crucially, it retained its electrochemical activity in sodium-based phosphate buffers. The RP microelectrodes displayed electrocatalytic reduction of hydrogen peroxide at 0 to -50 mV (vs Ag/AgCl). To fabricate biosensors on the RP microelectrodes, we used a sol-gel film electrodeposition technique to create ATP and hypoxanthine biosensors as examples of the methodology. These RP-mediated biosensors displayed excellent performance including the following: high selectivity against interferences such as 5HT, ascorbic acid, urate, and acetaminophen; high sensitivity with wide linear calibration range; and good stability. These attractive characteristics demonstrate that RP can be universally employed as an electron mediator in fabrication of highly selective oxidase-based microelectrode biosensors. Furthermore, given their ability to operate in the presence of physiological levels of Na+, the RP-mediated biosensors can be potentially applied to the in vitro and in vivo measurement of physiological signaling substances.     

Direct electrochemical detection of purine- and pyrimidine-based nucleotides with sinusoidal voltammetry

Sinusoidal voltammetry was employed to detect both purine- and pyrimidine-based nucleic acids. Adenine and cytosine, representing these two classes of nucleic acids, could be measured with submicromolar detection limits at a copper electrode under these conditions, where the sensitivity for adenine was much higher than that for cytosine. Detection limits for purine-containing nucleotides [e.g., adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP)] were on the order of 70-200 nM using this method. These detection limits are achieved for native nucleotides and are over 2 orders of magnitude lower than those found with UV absorbance detection. Submicromolar detection limits were also obtained for pyrimidine-based nucleotides, which could also be detected with high sensitivity due to the presence of a sugar backbone that is electroactive at the copper surface. This detector is not fouled by the nucleotides and may be used for the sensitive detection of analytes eluting continuously in a flowing stream, i.e., from a chromatography column or an electrophoresis capillary.     

Entrapment of fluorescent signaling DNA aptamers in sol-gel-derived silica

We report on the first successful immobilization of a DNA aptamer, in particular, a fluorescence-signaling DNA aptamer, within a sol-gel-derived matrix. The specific aptamer examined in this study undergoes a structural switch in the presence of adenosine triphosphate (ATP) to release a dabcyl-labeled nucleotide strand (QDNA), which in turn relieves the quenching of a fluorescein label that is also present in the aptamer structure. It was demonstrated that aptamers containing a complementary QDNA strand along with either a short complimentary strand bearing fluorescein (tripartite structure) or a directly bound fluorescein moiety (bipartite structure) remained intact upon entrapment within biocompatible sol-gel derived materials and retained binding activity, structure-switching capabilities, and fluorescence signal generation that was selective and sensitive to ATP concentration. Studies were undertaken to evaluate the properties of the immobilized aptamers that were either in their native state or bound to streptavidin using a terminal biotin group on the aptamer, including response time, accessibility, and leaching. Furthermore, signaling abilities were optimized through evaluation of different QDNA constructs. These studies indicated that the aptamers remained in a state that was similar to solution, with moderate leaching, only minor decreases in accessibility to ATP, and an expected reduction in response time due to diffusional barriers to mass transport of the analyte through the silica matrix. Entrapment of the aptamer also resulted in protection of the DNA against degradation from nucleases, improving the potential for use of the aptamer for in vivo sensing. This work demonstrates that sol-gel-derived materials can be used to successfully immobilize and protect DNA-based biorecognition elements and, in particular, DNA aptamers, opening new possibilities for the development of DNA aptamer-based devices, such as affinity columns, microarrays, and fiber-optic sensors.     

Simultaneous detection of pH changes and histamine release from oxyntic glands in isolated stomach

Real-time simultaneous detection of changes in pH and levels of histamine over the oxyntic glands of guinea pig stomach have been investigated. An iridium oxide pH microelectrode was used in a potentiometric mode to record the pH decrease associated with acid secretion when the sensor approached the isolated tissue. A boron-doped diamond (BDD) microelectrode was used in an amperometric mode to detect histamine when the electrode was placed over the tissue. Both sensors provided stable and reproducible responses that were qualitatively consistent with the signaling mechanism for acid secretion at the stomach. Simultaneous measurements in the presence of pharmacological treatments produced significant variations in the signals obtained by both sensors. As the H2 receptor antagonist cimetidine was perfused to the tissue, histamine levels increased that produced an increase in the signal of the BDD electrode whereas the pH sensor recorded a decrease in acid secretion as expected. Addition of acetylcholine (ACh) stimulated additional acid secretion detected with the pH microelectrode whereas the BDD sensor recorded the histamine levels decreasing significantly. This result shows that the primary influence of ACh is directly on the parietal cell receptors rather then the ECL cell receptors of the oxyntic glands. These results highlight the power of this simultaneous detection technique in the monitoring and diagnosis of physiological significant signaling mechanisms and pathways.     

Lanthanide Recognition: Monitoring of Praseodymium(III) by a Novel Praseodymium(III) Microsensor Based on N′-(Pyridin-2-Ylmethylene)Benzohydrazide

In this work, for the first time, we introduce a highly selective and sensitive praseodymium(III) microsensor. Npsila-(pyridin-2-ylmethylene)benzohydrazide (PBH) was used as a membrane-active component to prepare a highly sensitive Pr(III)-selective polymeric membrane microelectrode. The electrode exhibits a Nernstian response toward Pr(III) ions over a very wide concentration range (1.0 times 10^-3 -1.0 times 10^-8 M), with a detection limit of 7.0 times 10^-9 M (~1 ng/ml). It has a very fast response time in the whole concentration range (~10 s). The proposed microelectrode can be used for at least six weeks without any considerable divergence in potentials. The proposed membrane sensor revealed very good selectivity toward Pr(III) ions over a wide variety of other metal ions including common alkali, alkaline earth and, specially, lanthanide ions. It could be used in the pH range of 3.0-8.5. The Pr(III) microelectrode was used as an indicator electrode for the titration of 20 ml of a 1.0 times 10^-6 M Pr(III) ions with a 1.0 times 10^-4 M EDTA.     

Graphene enhanced electron transfer at aptamer modified electrode and its application in biosensing

Graphene (GN), a two-dimensional and one-atom thick carbon sheet, is showing exciting applications because of its unique morphology and properties. In this work, a new electrochemical biosensing platform by taking advantage of the ultrahigh electron transfer ability of GN and its unique GN/ssDNA interaction was reported. Adenosine triphosphate binding aptamer (ABA) immobilized on Au electrode could strongly adsorb GN due to the strong π-π interaction and resulted in a large decrease of the charge transfer resistance (R(ct)) of the electrode. However, the binding reaction between ABA and its target adenosine triphosphate (ATP) inhibited the adsorption of GN, and R(ct) could not be decreased. On the basis of this, we developed a new GN-based biosensing platform for the detection of small molecule ATP. The experimental results confirmed that the electrochemical aptasensor we developed possessed a good sensitivity and high selectivity for ATP. The detection range for ATP was from 15 × 10(-9) to 4 × 10(-3) M. The method here was label-free and sensitive and did not require sophisticated fabrication. Furthermore, we can generalize this strategy to detect Hg(2+) using a thymine (T)-rich, mercury-specific oligonucleotide. Therefore, we expected that this method may offer a promising approach for designing high-performance electrochemical aptasensors for the sensitive and selective detection of a spectrum of targets.     

Immobilization of purine bases on a poly-4-aminophenol matrix

Conducting polymers or semi-conductors have various features that make them excellent materials for the immobilization of biomolecules and the rapid transfer of electrons necessary for the production of biosensors. Conducting electroactive polymers of poly-4-aminophenol have been developed as sensors to detect the purine bases (adenosine triphosphate, ATP and guanosine triphosphate, GTP) of DNA. The electrooxidation of 4-aminophenol onto a graphite electrode in the presence of perchloric acid yielded thin polymer films. The conductivity was studied by cyclic voltammetry and surface morphology by optical microscopy and interferometry. The immobilization and detection of ATP and GTP on a graphite electrode or modified electrode coated with poly-4-aminophenol was studied by cyclic voltammetry. Systematic variation of the experimental conditions that influenced the electrode reaction, particularly the pH of the electrolytic solution, showed that the oxidation potentials of the immobilized ATP or GTP in the modified electrodes decreased with increasing pH of the electrolyte. When these conditions were optimized based on voltammetric measurements, modified electrodes coated with poly-4-aminophenol were found to be efficient in immobilizing purine bases, and increased the amplitude of the ATP and GTP signals by ∼1.5 and ∼24 times, respectively, when compared with non-coated graphite surfaces.     

Fiber-optic microsensors to measure backscattered light intensity in biofilms

We have constructed tapered fiber-optic microsensors with a tip diameter of less than 10 mum to measure profiles of backscattered light in biofilms, which are thin layers of micro-organisms firmly attached to surfaces. The observed response agrees well with local effective diffusivity microelectrode measurements, with R(2) > 0.85. A strong relation between signal intensity and wavelength has been observed at 670 and 1320 nm. These sensors have the potential to replace local effective diffusivity microelectrodes for true in situ biofilm measurements.     

Photo‐electrochemical Bioanalysis of Guanosine Monophosphate Using Coupled Enzymatic Reactions at a CdS/ZnS Quantum Dot Electrode

A photo‐electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)‐dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co‐substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co‐substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD⁺. This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.     

Multifunctional label-free electrochemical biosensor based on an integrated aptamer

Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A Au electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA). The unimolecular MBA contained small-molecule ATP binding aptamer (ABA) and also protein alpha-thrombin binding aptamer (TBA). Thus, the aptasensor could be used for detection of ATP and alpha-thrombin both. The detection limit of ATP was 1 x 10(-8) M, and its detection range could extend up to 10(-4) M, whereas the detection limit of alpha-thrombin was 1 x 10(-11) M, and its detection range was from 1 x 10(-11) to 1 x 10(-7) M. Meanwhile, after detecting alpha-thrombin, the sensing interface could be used for ATP recognition as well. The aptasensor regeneration could be realized by rehybridizing of the MBA strand with the partly complementary strand immobilized on the Au surface after ATP detection or by treating with a large amount of ATP and then rehybridizing the MBA strand with the partly complementary strand immobilized on the Au surface after alpha-thrombin detection. The aptasensor fabricated exhibited several advantages such as label-free detection, high sensitivity, regeneration, and multifunctional recognition. It also showed the detectability in biological fluid. Therein it held promising potential for integration of the sensing ability such as the simultaneous detection for multianalysis in the future.     

Reusable amperometric biosensor for measuring protein tyrosine kinase activity

This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.     

Highly sensitive electrogenerated chemiluminescence biosensor in profiling protein kinase activity and inhibition using gold nanoparticle as signal transduction probes

A novel electrogenerated chemiluminescence (ECL) biosensor using gold nanoparticles as signal transduction probes was described for the detection of kinase activity. The gold nanoparticles were specifically conjugated to the thiophosphate group after the phosphorylation process in the presence of adenosine 59-[c-thio] triphosphate (ATP-s) cosubstrate. Due to its good conductivity, large surface area, and excellent electroactivity to luminol oxidization, the gold nanoparticles extremely amplified the ECL signal of luminol, offering a highly sensitive ECL biosensor for kinase activity detection. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of 0.07 U mL(-1), wide linear range (from 0.07 to 32 U mL(-1)), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC(50) of ellagic acid, a PKA inhibitor, was estimated, which was in agreement with those characterized with the conventional kinase assay. While nearly no ECL signal change can be observed in the presence of Tyrphostin AG1478, a tyrosine kinase inhibitor, but not PKA inhibitor, shows its excellent performance in kinase inhibitor screening. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.     

Amplified fluorescence of Mg2+ selective red-light emitting carbon dot in water and direct evaluation of creatine kinase activity

Carbon quantum dot/carbon dot (CD) exhibiting sustained photoluminescence at longer wavelengths in aqueous solution is difficult to prepare, but has enormous potential in biomedical applications. For the first time, we report the magnesium(II) selective fluorescence enhancement of a red-light emitting anthrarufin and boric acid-derived CD in aqueous solution for direct evaluation of creatine kinase (CK) enzyme activity. The CD displayed visually detectable, intense red fluorescence only in the presence of magnesium ion (Mg²⁺) at physiological pH value when irradiated with an ultraviolet (UV) source. Concurrently, a significant increase in steady-state fluorescence intensity and fluorescence lifetime was documented. A time-dependent density functional theory (TD-DFT) analysis displayed a bathochromic shift in UV-visible (vis) absorption, and increased oscillator strength of transition resulting from the selective chelation of Mg²⁺ with β-hydroxy keto functionality on the surface of the CD. The CD-Mg²⁺ assembly was subsequently used to conceptualize the detection of CK directly through the exploration of the differential binding affinity of Mg²⁺ with adenosine triphosphate (ATP), adenosine diphophate (ADP), and CD that is otherwise not possible with commercially available kits as of today. Thus, the report delineated here usher grandeur potential of CD for biological explorations related to Mg²⁺ or ATP sensing and monitoring of Mg²⁺-dependent enzymatic activity through a clear understanding of the chemistry.

     

Highly sensitive pyrosequencing based on the capture of free adenosine 5' phosphosulfate with adenosine triphosphate sulfurylase

In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, adenosine triphosphate (ATP) sulfurylase, luciferase, and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes a photoemission reaction. However, the side-reaction of adenosine 5' phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence, the sensitivity cannot be much higher due to the simultaneous increase of the background signal when a larger amount of luciferase is used. In this study, we demonstrated a sensitive pyrosequencing using a large amount of ATP sulfurylase to lower the concentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase, a large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencing chemistry allows the use of an inexpensive light sensor photodiode array for constructing a portable pyrosequencer, a potential tool in a point-of-care test (POCT).