Adenosine In Serum Research Articles

A novel “off-on” ratiometric fluorescent aptasensor for adenosine detection based on FRET between quantum dots and graphene oxide

A novel “off-on” ratiometric fluorescent aptasensor was established for adenosine detection based on fluorescence resonance energy transfer (FRET) between CdS QDs, DNA QDs as donor and graphene oxide (GO) as acceptor. Amino-riched DNA QDs covalently bonded to the carboxyl group on the edge of the GO, and with the absorption of the TGA-modified CdS QDs with aptamer (CdS QDs-apt) onto the GO surface via the π-π stacking interaction. The fluorescence of both CdS QDs and DNA QDs were efficiently quenched due to FRET (turn off). When adenosine was present, the specific binding of the aptamer to the target preferentially that released the CdS QDs-apt from GO. The process would inhibit the FRET which contribute to the fluorescence of CdS QDs-apt recovery again (turn on), while the fluorescence intensity of DNA QDs only slightly altered and acted as the reference signal. Thus, a novel “off-on“ ratiometric fluorescent aptasensor for adenosine detection was constructed accordingly. There was a good linearity relationship between the ratio of the FL intensity (F595 nm/F464 nm) and the concentration of adenosine in the range of 20.00–180.0 nmol/L with a detection limit of 1.3 nmol/L (S/N = 3, n = 9). Importantly, the feasibility of the developed aptasensor for selective detection of adenosine in serum and urine samples with satisfactory results. The recoveries were observed to be 97.04–100.2 %.

Real-Time Tunable Dynamic Range for Calibration-Free Biomolecular Measurements with a Temperature-Modulated Electrochemical Aptamer-Based Sensor in an Unprocessed Actual Sample.

The sensing technologies for monitoring molecular analytes in biological fluids with high frequency and in real time could enable a broad range of applications in personalized healthcare and clinical diagnosis. However, due to the limited dynamic range (less than 81-fold), real-time analysis of biomolecular concentration varying over multiple orders of magnitude is a severe challenge faced by this class of analytical platforms. For the first time, we describe here that temperature-modulated electrochemical aptamer-based sensors with a dynamically adjustable calibration-free detection window could enable continuous, real-time, and accurate response for the several-hundredfold target concentration changes in unprocessed actual samples. Specifically, we could regulate the electrode surface temperature of sensors to obtain the corresponding dynamic range because of the temperature-dependent affinity variations. This temperature modulation method relies on an alternate hot and cold electrode reported by our group, whose surface could actively be heated and cooled without the need for altering ambient temperature, thus likewise applying for the flowing system. We then performed dual-frequency calibration-free measurements at different interface temperatures, thus achieving an extended detection window from 25 to 2500 μM for procaine in undiluted urine, 1-500 μM for adenosine triphosphate, and 5-2000 μM for adenosine in undiluted serum. The resulting sensor architecture could drastically expand the real-time response range accessible to these continuous, reagent-less biosensors.

P.353 Long‐acting injectable paliperidone as maintenance treatment for bipolar disorders

In this study, a sensitive electrochemical voltammetry method for the analysis of extra cellular adenosine (AD) using a carbon paste electrochemical (CPE) modified with a porous material, such as natural phosphate (NP), was proposed. Adenosine strongly adsorbed on a electrode surface and prepared electrode NP-CPE provides easy methods for the detection of low concentrations of extracellular adenosine using cyclic voltammetry (cv) and square wave voltammetry (swv). Some electrochemical properties, in particular the influence of pH, the effect of the electrolyte have been studied. The elaborate electrode was then subjected to an analytical application for the detection of extracellular adenosine in serum.

An in-tube aptamer/gold nanoparticles coated capillary solid-phase microextraction for separation of adenosine in serum and urine samples

As an endogenous nucleoside, adenosine was significant for the diagnosis and treatment of some diseases, such as schizophrenia. However, due to the complicated matrix interference, it was difficult to monitor trace or ultra-trace adenosine directly in bio-samples. In this contribution, a novel in-tube SPME technique based on aptamer/Au nanoparticles coated open tubular fused-silica capillary was established to separate and enrich adenosine in bio-samples with high affinity. Therefore, a uniform and dense AuNPs layer was coated on the inner surface of the open tubular capillary, and then adenosine aptamer was immobilized on AuNPs with a high capacity of 2.44 μg per 27-cm capillary. As a result, the capillary shown high selectivity to adenosine with a selectivity factor of 14.4 when compared with the scrambled aptamer/AuNPs coated capillary. Also, the extraction amount of adenosine was 2.8–24.8 times higher than those of its structural analogs and contrast, such as guanosine, uridine, cytidine, thymidine, and toluic acid. After the optimization of extraction conditions, the aptamer/AuNPs coated in-tube SPME-HPLC method was developed for the adenosine assay with the linear range of 0.002–0.100 μg mL−1 and the detection limit of 0.45 ng mL−1. Subsequently, the approach was applied for trace adenosine monitoring in human serum and urine samples. It showed a strong performance of reducing matrix interference and improving sensitivity, and the spiking recoveries of 89.9–92.6% and 91.1–94.5% were achieved respectively.

Electrochemical determination of adenosine by natural phosphate modified carbon paste electrode: Analytical application in serum

In this study, a sensitive electrochemical voltammetry method for the analysis of extra cellular adenosine (AD) using a carbon paste electrochemical (CPE) modified with a porous material, such as natural phosphate (NP), was proposed. Adenosine strongly adsorbed on a electrode surface and prepared electrode NP-CPE provides easy methods for the detection of low concentrations of extracellular adenosine using cyclic voltammetry (cv) and square wave voltammetry (swv). Some electrochemical properties, in particular the influence of pH, the effect of the electrolyte have been studied. The elaborate electrode was then subjected to an analytical application for the detection of extracellular adenosine in serum.

Serum and brain purine levels in an experimental systemic infection of mice by Cryptococcus neoformans: Purinergic immunomodulatory effects

The aim of this study was to evaluate the purine levels in serum and brains of mice experimentally infected by Cryptococcus neoformans. Twenty-four mice were divided into the following groups: a control group (n = 12; Group A) and an infection group with animals that were infected (n = 12; Group B) with a 0.3-mL intraperitoneal injection containing 1.7 × 107C. neoformans cells. Blood and brains were collected on days 20 (n = 6 per group) and 50 (n = 6 per group) post-infection (PI). Histopathology and lung and brain cultures were performed to confirm fungal infection and tissue injuries. The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (HYPO), xanthine (XAN) and uric acid (UA) in brains and serum were measured by high-performance liquid chromatography (HPLC) analysis. At both time points, histopathology analysis revealed inflammatory infiltrates in the brains and lungs of infected mice; clinical signs, such as piloerection and clinical respiratory distress, were also observed. ATP levels were significantly increased on days 20 and 50 PI (P < 0.01) in brains and serum, while brain ADO levels were increased on day 20 PI; brain and serum ADO levels were decreased on day 50 PI. Levels of ADP and AMP did not differ between groups (P > 0.05). Serum levels of INO of infected mice increased only on day 50 PI (P < 0.05). HYPO levels were reduced in the brains of infected animals at both experimental time points and were decreased in serum at day 50 PI (P < 0.05). XAN levels increased in infected mice only in serum on day 50 PI (P < 0.05). The endogenous anti-oxidant uric acid was significantly increased in brain (days 20 and 50 PI) and decreased in serum. It is possible that C. neoformans infection in mice leads to a high ATP/ADO ratio that may improve the brain pro-inflammatory response during both periods, while high ATP levels in serum act as a systemic signal to improve the immune response. Moreover, the anti-oxidant uric acid may increase in the brain to protect inflamed tissue from oxidative stress.

A target triggered proximity combination-based fluorescence sensing strategy for adenosine detection.

Adenosine is a potent physiological and pharmacological regulator, and its abnormal level is closely related to disease development. The sensitive and specific detection of adenosine is crucial for health evaluation and disease diagnosis. In this work, a target triggered proximity combination-based fluorescence sensing strategy is developed for the sensitive and specific detection of adenosine. A difunctional probe showing target recognition and signal amplification is designed, by integration of DNA linker-connected split aptamer fragments with a fragment-elongated polymerase/nicking template. The presence of adenosine would glue the split aptamers, which triggers the two distal aptamer fragments to combine with each other into proximity. The approaching aptamer fragment ends then initiate the strand displacement amplification (SDA) reaction, generating numerous DNA primers. The DNA primers further hybridize with a padlock probe and initiate the rolling circle amplification (RCA) reaction, producing numerous G-quadruplex sequences. The G-quadruplex sequences finally bind with Thioflavin T to obtain enhanced fluorescence signals. The method exhibits a linear correlation within the adenosine concentration range from 5.0 × 10-7 M to 2.0 × 10-5 M (R = 0.999) with a detection limit of 8.4 × 10-8 M, and a good selectivity to distinguish adenosine from its analogues. The recoveries of adenosine in human serum are from 91% to 94%, demonstrating that the system works well in biological fluids. The proposed sensing strategy is anticipated to hold promise in biochemical research, clinical diagnosis and disease treatment.

Graphene/Cu nanoparticle hybrids fabricated by chemical vapor deposition as surface-enhanced Raman scattering substrate for label-free detection of adenosine.

We present a graphene/Cu nanoparticle hybrids (G/CuNPs) system as a surface-enhanced Raman scattering (SERS) substrate for adenosine detection. The Cu nanoparticles wrapped by a monolayer graphene shell were directly synthesized on flat quartz by chemical vapor deposition in a mixture of methane and hydrogen. The G/CuNPs showed an excellent SERS enhancement activity for adenosine. The minimum detected concentration of the adenosine in serum was demonstrated as low as 5 nM, and the calibration curve showed a good linear response from 5 to 500 nM. The capability of SERS detection of adenosine in real normal human urine samples based on G/CuNPs was also investigated and the characteristic peaks of adenosine were still recognizable. The reproducible and the ultrasensitive enhanced Raman signals could be due to the presence of an ultrathin graphene layer. The graphene shell was able to enrich and fix the adenosine molecules, which could also efficiently maintain chemical and optical stability of G/CuNPs. Based on the G/CuNPs system, the ultrasensitive SERS detection of adenosine in varied matrices was expected for the practical applications in medicine and biotechnology.

High Phosphate Serum Levels Correlate With the Severity of Experimental Severe Acute Pancreatitis

Extracellular purines are a component of the systemic inflammatory response, and their levels are modulated by ectonucleotidases. In addition, nucleotide hydrolysis releases phosphate. We studied serum phosphate levels as a predictor of severity in acute pancreatitis (AP) and their correlation with extracellular purinergic metabolism. Acute pancreatitis was induced by the retrograde injection of sodium taurocholate. The AP group was compared with animals submitted to a model of sepsis by cecal ligation and puncture. The sham group was submitted to laparotomy and closure. We measured the phosphate and purine levels in serum and the expression of 5'-nucleotidase (CD73) and the adenosine A2a receptor in pancreatic tissue by quantitative real-time polymerase chain reaction. Serum phosphate levels were higher in severe AP and correlated with severity. Severe AP led to increased serum levels of adenosine diphosphate, adenosine monophosphate, and adenosine. In addition, adenosine monophosphate conversion to adenosine in serum was accelerated in the AP groups. We found a positive correlation between serum adenosine and phosphate in the AP groups. The expression levels of CD73 and the adenosine A2a receptor in the pancreas were not altered. Our study suggests that serum phosphate correlates with severity in AP and implicates extracellular purines in the systemic response to severe AP.

Effect of Sustaining Dietary Ketosis on the Hippocampal and Serum Metabolome of Sprague‐Dawley Rats

The ketogenic diet (KD) has been successfully used to treat pediatric refractory epilepsy since the 1920s and is currently being investigated as an adjunct treatment for many disease states. The therapeutic efficacy of the KD results from an elevation of blood ketones. We previously reported that oral administration of ketone supplements produced nutritional ketosis (>0.5 mM) without carbohydrate restriction and the effects of a 28-day administration of five ketone supplements on blood glucose, ketones, and lipids in male Sprague-Dawley rats. We hypothesized that the 28-day administration would affect metabolomic markers. Serum (~300 μL) and hippocampal tissues for ketone supplements 1,3 butanediol acetoacetate diester (KE) and (1:1 mixture of sodium/potassium beta-hydroxybutyrate mineral salt solution (BMS) and medium chain triglyceride oil (MCT)) BMS+MCT were collected on day 28 at 4 hours post-intragastric gavage (peak ketone elevation). Metabolon analyzed samples by gas and liquid chromatography in tandem with mass spectrometry. Both ketone supplements significantly increased serum TCA cycle intermediates: citrate, fumarate and malate. KE supplement significantly increased alpha-ketoglutarate and BMS+MCT supplement significantly increased succinate in the rat serum. Additionally, both supplements affected medium chain fatty acids, dicarboxylic acids, adenosine, inflammatory mediators, antioxidants, and bile acids in both serum and hippocampal tissue. Although both forms of ketone supplementation increased brain and blood ketone levels, the global metabolic profiles were different.

A general strategy based on luminescent oxygen channeling for the detection of adenosine in serum using the steric hindrance effect of thrombin

A major challenge in the field of biosensing is high-throughput screening (HTC) of small molecules in complex biological media such as human serum. Two aspects are required to achieve the goal: (1) General assays for target-independent detection of different molecules specifically and sensitively; (2) the operation is able to be standardized with automation possibility. This work integrates the distinguishing features of aptamer and fluorescent techniques. Innovatively, a very easy method is designed to combine a long range diffusion triggered excitation and a great steric hindrance effect, which makes it possible to become a general sensing method. As a proof-of-principle, adenosine in undiluted serum was detected. A nice detection limit and linear relationship were obtained.

Alterations in the cholinesterase and adenosine deaminase activities and inflammation biomarker levels in patients with multiple sclerosis

Multiple sclerosis (MS) is one of the main chronic inflammatory diseases of the CNS that cause functional disability in young adults. It has unknown etiology characterized by the infiltration of lymphocytes and macrophages into the brain. The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes and whole blood, as well as butyrylcholinesterase (BChE) and adenosine deaminase (ADA) activities in serum. We also checked the levels of nucleotides, nucleosides, biomarkers of inflammation such as cytokines (interleukin (IL)-1, IL-6, interferon (IFN)-γ, tumor necrosis factor-alpha (TNF-α) and IL-10) and C-reactive protein (CRP) in serum from 29 patients with the relapsing-remitting form of MS (RRMS) and 29 healthy subjects as the control group. Results showed that AChE in lymphocytes and whole blood as well as BChE, and ADA activities in serum were significantly increased in RRMS patients when compared to the control group (P<0.05). In addition, we observed a decrease in ATP levels and a significant increase in the levels of ADP, AMP, adenosine and inosine in serum from RRMS patients in relation to the healthy subjects (P<0.05). Results also demonstrated an increase in the IFN-γ, TNF-α, IL-1, IL-6 and CRP (P<0.05) and a significant decrease in the IL-10 (P<0.0001) in RRMS patients when compared to control. Our results suggest that alterations in the biomarkers of inflammation and hydrolysis of nucleotides and nucleosides may contribute to the understanding of the neurological dysfunction of RRMS patients.

A new method for the detection of adenosine based on time-resolved fluorescence sensor

In this work, we report a thrombin binding aptamer complex based time-resolved fluorescence sensor for small molecule detection. The sensor employs two strands (DNA1 and DNA2) of oligonucleotides. This two strands of oligonucleotides contain two aptamer (α-aptamer and β-aptamer) respectively. DNA1 and DNA2 were labeled with biotin and DIG at the 3′-end, respectively. Binding of the α-aptamer and β-aptamer to the thrombin promotes the hybridization between the complementary stem sequences attached to the two oligonucleotide sequences. The hybridization then brings biotin to be hidden in the shield part on DNA1, shielding biotin from being approached by the streptavidin modified on the microplate due to the steric hindrance effect of the shield part of DNA1. Result in the thrombin–aptamer complex cannot be modified on the surface of microplate which further leads to no signal reported. The strategy integrates the distinguishing features of aptamer and fluorescent techniques. As a proof-of-principle, adenosine in serum was detected with a detection limit of 0.5nM. A nice detection limit and linear relationship were obtained.

Hollow core photonic crystal fiber as a reusable Raman biosensor

We report that a single hollow core photonic crystal fiber (HC-PCF) can be used for repetitive characterization of multiple samples by Raman spectroscopy. This was achieved by integrating the HC-PCF to a differential pressure system that allowed effective filling, draining and re-filling of samples into a HC-PCF under identical optical conditions. Consequently, high-quality and reliable spectral data could be obtained which were suitable for multivariate analysis (partial least squares). With the present scheme, we were able to accurately predict different concentrations of heparin and adenosine in serum. Thus the detection scheme as presented here paves a path for the inclusion of HC-PCFs in point-of-care technologies and environmental monitoring where rapid sample characterization is of utmost importance.

Direct detection of adenosine in undiluted serum using a luminescent aptamer sensor attached to a terbium complex.

Aptamers, single-stranded nucleic acids that can selectively bind to various target molecules, have been widely used for constructing biosensors. A major challenge in this field, however, is direct sensing of analytes in complex biological media such as undiluted serum. While progress has been made in developing an inhomogeneous assay by using a preseparation step to wash away the interferences within serum, a facile strategy for direct detection of targets in homogeneous unprocessed serum is highly desired. We herein report a turn-on luminescent aptamer biosensor for the direct detection of adenosine in undiluted and unprocessed serum, by taking advantage of a terbium chelate complex with long luminescence lifetime to achieve time-resolved detection. The sensor exhibits a detection limit of 60 μM adenosine while marinating excellent selectivity that is comparable to those in buffer. The approach demonstrated here can be applied for direct detection and quantification of a broad range of analytes in biological media by using other aptamers.

Influence of Trypanosoma evansi in adenine nucleotides and nucleoside concentration in serum and cerebral cortex of infected rats

This study aimed to evaluate the adenine nucleotides and nucleoside concentration in serum and cerebral cortex of rats infected with Trypanosma evansi. Each rat was intraperitoneally infected with 1×106 trypomastigotes suspended in cryopreserved blood (Group A; n=18). Twelve animals were used as controls (Group B). The infected animals were monitored daily by blood smears. At days 4 and 20 post-infection (PI) it was collected serum and cerebral cortex to measure the levels of ATP, ADP, AMP and adenosine by high performance liquid chromatography (HPLC). In serum there was a significant (P<0.05) increase in the ATP, AMP and adenosine concentrations at days 4 and 20 PI in infected rats when compared to not-infected. Furthermore, in the cerebral cortex it was observed a significant (P<0.05) increase in the concentrations of ATP, AMP and decreased adenosine levels at day 4 PI. At day 20 PI it was only observed an increase in the AMP and adenosine concentrations in cerebral cortex of infected rats when compared to not-infected. It was not observed any difference in ADP concentration in serum and brain at days 4 and 20 PI. No change was observed histologically in the cerebral cortex of infected animals. The results allow us to conclude that infection with T. evansi in rats causes an increase in the concentrations of ATP, AMP and adenosine in serum and cerebral cortex the time periods evaluated. These alterations occurred as a result of T. evansi infection which involves neurotransmission, neuromodulation and immune response impairment confirm the importance of the purinergic system in this pathology.

Base Pairing at the Abasic Site in DNA Duplexes and Its Application in Adenosine Aptasensors

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 μM for adenosine and 41.8 μM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 μM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.

Simultaneous LC–UV–MS–MS Analysis of Nine Pivotal Metabolites in Human Serum: Application to Studies of Impaired Glucose Tolerance

A rapid, sensitive, and selective LC–UV–MS–MS method for simultaneous quantification of uric acid, creatinine, xanthine, creatine, hypoxanthine, adenosine, inosine, thymidine, and uridine in serum from patients with impaired glucose tolerance (IGT) has been developed and validated. After precipitation of protein in the serum with methanol samples were evaporated with a stream of nitrogen then reconstituted with aqueous ammonia (0.002 mol L−1). Chromatographic separation was performed on a C18 column with methanol–buffer (8 mmol L−1 ammonium acetate adjusted to pH 6.1 with glacial acetic acid) as mobile phase at a flow rate of 0.8 mL min−1. UV and MS–MS detection were used to quantify the metabolites on the basis of different concentrations and detector response. Linearity was excellent, with r 2 no 85, <10, and <10%, respectively. This reliable bioanalytical method enables evaluation of the levels of purines and pyrimidines in serum for IGT studies and diagnosis.

Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor

Aptamers are single-stranded nucleic acids that can selectively bind to essentially any molecule of choice. Because of their high stability, low cost, ease of modification, and availability through selection, aptamers hold great promise in addressing key challenges in bioanalytical chemistry. In the past 15 years, many highly sensitive fluorescent aptamer sensors have been reported. However, few such sensors showed high performance in serum samples. Further challenges related to practical applications include detection in a very small sample volume and a low dependence of sensor performance on ionic strength. We report the immobilization of an aptamer sensor on a magnetic microparticle and the use of flow cytometry for detection. Flow cytometry allows the detection of individual particles in a capillary and can effectively reduce the light scattering effect of serum. Since DNA immobilization generated a highly negatively charged surface and caused an enrichment of counterions, the sensor performance showed a lower salt dependence. The detection limits for adenosine are determined to be 178 microM in buffer and 167 microM in 30% serum. Finally, we demonstrated that the detection can be carried out in 10 microL of 90% human blood serum.

Involvement of Adenosine in the Antiinflammatory Action of Ketamine

Ketamine is an anesthetic drug. Subanesthetic doses of ketamine have been shown to reduce interleukin-6 concentrations after surgery and to reduce mortality and the production of tumor necrosis factor alpha and interleukin 6 in septic animals. Similarly, adenosine was shown to reduce tumor necrosis factor alpha and mortality of septic animals. The aim of this study was to determine whether adenosine mediates the antiinflammatory effects of ketamine. Sepsis was induced in mice by lipopolysaccharide or Escherichia coli inoculation. Leukocyte recruitment and cytokine concentrations were used as inflammation markers. Adenosine concentrations were assayed by high-performance liquid chromatography, and the involvement of adenosine in the effects of ketamine was demonstrated by adenosine receptor agonists and antagonists. Ketamine markedly reduced mortality from sepsis, leukocyte recruitment, and tumor necrosis factor-alpha and interleukin-6 concentrations. Ketamine administration in mice and rats was associated with a surge at 20-35 min of adenosine in serum (up to 5 microm) and peritoneal fluid. The adenosine A2A receptor agonist CGS-21680 mimicked the effect of ketamine in peritonitis, whereas the A2A receptor antagonists DMPX and ZM 241385 blocked its antiinflammatory effects. In contrast, A1 and A3 receptor antagonists had no effect. ZM 241385 reversed the beneficial effect of ketamine on survival from bacterial sepsis. The current data suggest that the sepsis-protective antiinflammatory effects of ketamine are mediated by the release of adenosine acting through the A2A receptor.