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New Strategy to Preserve Phosphate by Ionic Liquid Matrices in Matrix-Assisted Laser Desorption/Ionization: A Case of Adenosine Nucleotides
摘要： Adenosine -5'-triphosphate (ATP) plays a valuable role in metabolic activity to produce adequate energy in a biosystem. A high ATP/AMP ratio has a correlation with diabetes that induces suppression of AMP-activated protein kinase (AMPK). Matrix-assisted laser desorption/ionization (MALDI)–mass spectrometry (MS) has outstanding potential in determining the ratio of several types of adenosine phosphates in a sample to rapidly understand the primary energy transfer in metabolism. Although MALDI is viewed as a soft ionization technique for MS analysis, excess photon energy might crack the phosphate bonds leading to misinterpretation of the ATP level. In this work, ionic liquid matrices (ILMs) were employed to reduce fragmentation and increase the detection e?ciency during the MALDI process. This study demonstrated for the ?rst time that 2,5-dihydroxybenzoic acid pyridine (DHBP) is one of the most e?ective matrices for further quantitative analysis of adenosine nucleotides. This systematic screening of ILMs also enhances the fundamental understanding of MALDI.
关键词： Adenosine,ionic liquid matrix,AMPK,triphosphate,soft ionization,ionic liquids,ATP,nucleotides,MALDI
更新于2025-09-23 15:19:57
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Single-Cell ATP Content Monitoring during Hyperthermia Cell Death by using Plasmonic Fluorescent Nanoflare
摘要： Gold nanorods-based plasmonic photothermal therapy (AuNRs-PPTT) has been a prospective anti-cancer approach in which AuNRs absorbs near-infrared (NIR) light and converts it into heat, leading to cell death. Investigating molecular energy metabolism of single cells, especially cancer cells, during hyperthermia cell death process is therefore of great significance, as it can help us to better understand the photothermal lethal mechanism of cancer cells and design new photothermal probes more rationally. However, during the AuNRs-PPTT process, how the cells respond to heat stimulation, and how their energy metabolism changes, these basic issues have rarely been studied. Herein, we selected adenosine triphosphate (ATP) as a target molecule, and by preparing a plasmonic and turn-on type fluorescent nanoprobe we examined the ATP metabolism difference between cancerous cells and normal cells during the AuNRs-PPTT process. We found that the fluorescence intensity increased ~ 60% after 5 min laser irradiation as compared to the initial intensity in single HeLa cells, but only ~ 20% increasement was observed for single H8 cells; obviously the increase of ATP content in cancerous cells was notably higher than that in normal cells during the hyperthermia cell death.
关键词： Plasmonic photothermal therapy,Fluorescent nanoprobe,ATP,Gold nanorods,Hyperthermia cell death
更新于2025-09-19 17:13:59
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Novel fluorescently labelled ATP analogues for direct monitoring of ubiquitin activation
摘要： Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed novel fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear – in contrast to earlier reports – only a single acridone-based dye at the terminal phosphate group. The dye’s fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.
关键词： ubiquitin,fluorescent probes,PET,UBA1,ATP
更新于2025-09-19 17:13:59
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Green fluorescent carbon quantum dots functionalized with polyethyleneimine, and their application to aptamer-based determination of thrombin and ATP
摘要： Brightly fluorescent carbon quantum dots coated with polyethylenimine (PEI-CDs) were prepared using malic acid and PEI as the precursors. The PEI-CDs have a high quantum yield (41%) and green emission (peaking at 502 nm under 430 nm excitation), both of which are not affected by high ionic strength. The PEI-CDs have a positive charge at physiological pH values and can electrostatically bind aptamers with their negative charge. This is shown for aptamers binding thrombin or ATP. Binding of aptamers results in quenching of fluorescence. If thrombin or ATP are introduced, the respective aptamer will bind them, and the complex is then released from the PEI-CDs. Fluorescence increases in proportion to the analyte concentration. Under optimized conditions, thrombin and ATP can be sensitively and selectively detected by fluorometry with lower detection limits of 1.2 and 13 nM, respectively. The assay was successfully applied to the determination of thrombin and of ATP in spiked serum samples.
关键词： Aptamer sensor,Thrombin detection,Serum analysis,Green luminescent,PEI-CDs,Malic acid,Polyethylenimine,Fluorescence assay,ATP detection,Hydrothermal reaction
更新于2025-09-19 17:13:59
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Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification
摘要： Abnormal concentration of adenosine triphosphate (ATP) is directly asscociate with several diseases. Thus, sensitive detection of ATP is essential to early diagnosis of disease. Herein, we described an ultrasensitive strategy for ATP detection by using positively charged gold nanorods ((+)AuNPs) as an efficient fluorescence quenching platform, coupled with exonuclease (cid:1) (Exo (cid:1)) assisted target recycling amplification. To construct the sensor, DNA template that contained ATP aptamer was used for the formation of AgNCs signal probe (DNA/AgNCs), the structure of it could change to duplex after the interaction of it with ATP. Such DNA template or duplex DNA product could electrostatically adsorb onto (+)AuNRs surface, resulting in the quenching of the fluorescence signal due to the vicinity of AgNCs to (+)AuNRs. With the addition of Exo (cid:1), DNA duplex could be hydrolyzed and released from (+)AuNRs surface, leading to the recovery of a strong fluorescent signal, while ATP could be regenerated for next target recycling. Combing the good fluorescence quenching ability of (+)AuNRs and the Exo (cid:1) assisted signal amplification, a low detection limit of 26 pM was achieved for ATP detection. Notably, the proposed method can be successfully applied for detecting ATP in serum samples, indicating a potential application value in early cancer diagnosis.
关键词： Exo (cid:1),Fluorescent sensor,(+)AuNRs,DNA/AgNCs,ATP detection,Target recycling amplification
更新于2025-09-16 10:30:52
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An enzyme-free FRET nanoprobe for ultrasensitive ketamine detection based on ATP-fueled target recycling
摘要： Ketamine is a commonly abused drug due to its stimulant, dissociative and hallucinogenic effects. An overdose of ketamine has been found to cause a variety of side effects. Therefore, the identification and quantification of ketamine are of significant importance for clinical purposes and drug seizing. However, conventional methods for ketamine detection possess some disadvantages such as sophisticated procedures, expensive instruments and low sensitivity. Herein, we develop a novel fluorescent nanoprobe for ultrasensitive ketamine detection with signal amplification based on Adenosine Triphosphate (ATP)-fueled target recycling and FRET (fluorescence resonance energy transfer) occurring between the FAM (Fluorescein, tagged with Y-shape DNA) and AuNPs. Based on the combination of FRET and signals circle amplification, the gold nanospheres functionalized with Y-motif DNA (Y@AuNPs) nanoprobe was utilized for effective ketamine detection with the limit of detection (LOD) down to 3 pg mL?1, which was lower than previously reported. Furthermore, the high sensitivity of Y@AuNPs facilitated quantitative analysis in biological media and practical samples.
关键词： Ultrasensitive detection,Ketamine,FRET,ATP-fueled target recycling,Fluorescent nanoprobe
更新于2025-09-16 10:30:52
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Low-Level Laser Therapy with a 635 nm Diode Laser Affects Orthodontic Mini-Implants Stability: A Randomized Clinical Split-Mouth Trial
摘要： Background: The study aimed to clinically estimate an influence of a 635 nm diode laser on the stability of orthodontic mini-implants, to assess mini-implants loss, and to evaluate a pain level after the treatment. Materials and Methods: The randomized clinical split-mouth trial included 20 subjects (13 women and 7 men; age: 32.5 ± 6.1 years), 40 implants (RMO, West Colfax Ave., Denver, CO, USA) with a diameter 1.4 mm and length of 10 mm. Mini-implants were placed in the area of the attached gingiva between the second premolar and first molar teeth, 2 mm below the mucogingival junction of both sides of the maxilla. Each implant on the right side (G1, n = 20) of the maxilla was irradiated with a diode laser, and the implants on the opposite side (left, G2, n = 20) were a control group (without laser irradiation). The 635-nm laser parameters; dose: 10 J per point (20 J/cm2), time: 100 s per point, two points (irradiation on a buccal, and a palatal side of the alveolus/implant), the total energy per session 20 J. Laser application protocol: immediately and 3, 6, 9, 12, 15, and 30 days after surgery. The total energy after all therapeutic sessions was 140 J. The implants’ stability was measured employing a Periotest device (Periotest Test Value—PTV) immediately and 3, 6, 9, 12, 15, 30, and 60 days after the insertion of the mini-implants. Results: We found significantly higher secondary stability, lower mean PTV (6.18 ± 5.30) and (1.51 ± 2.25), for self-drilling mini-implants (G1, test group) in contrast to the control, G2 group (9.17 ± 8.25) and (5.00 ± 3.24), after 30 (p = 0.0003) and 60 days (p = 0.0000). Moreover, the analysis of the mini-implants stability after 635-nm diode laser application revealed significant higher stability in comparison with none irradiated implants (G2 group) after 3 days. (p = 0.0000) There was no significant difference in pain level measured on the NRS-11 scale on both sides of the maxilla. (p = 0.3665) An important finding was that all inserted mini-implants survived during a two-month observation period. Conclusions: 635-nm diode laser at laser irradiation increases the secondary stability of orthodontic mini-implants.
关键词： biostimulation,periotest,micro-screw,semiconductor laser,ATP
更新于2025-09-16 10:30:52
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pH-Controlled Intracellular in Situ Reversible Assembly of Photothermal Agent for Smart Chemo-Photothermal Synergetic Therapy and ATP Imaging
摘要： To advance anti-tumor efficiency and lessen adverse effect caused by nanodrug residues in body, a smart nanoagent system is developed and successfully used in intracellular ATP-imaging and in vivo chemo-photothermal synergetic therapy. The nanoagent system is facilely prepared using DNA complex to modify gold nanoparticles (AuNPs). The DNA complex is formed by three oligonucleotides (ATP aptamer, rC-DNA and rG-DNA). CG-rich structure in ternary DNA complex could be exploited for payload of chemotherapeutic medicine doxorubicin (DOX), thus making efficient DOX transport into tumor site possible. In tumor cells, especially in acidic organelles (e.g. endosome and lysosome), DOX could be rapidly released via the dual stimuli of over-expressed ATP and pH. What’s more, the specific recognition of fluorescent-labelled aptamer strand to ATP can achieve the intracellular ATP imaging. pH-controlled reversible folding and unfolding of intermolecular i-motif formed by C-rich strands can lead to intracellular in situ assembly of AuNP aggregates with high photothermal conversion efficiency, and promote relatively facile renal clearance of AuNPs through the disassociation of the aggregates in extracellular environments. Experiments in vivo and vitro present feasibility for synergetic chemo-photothermal therapy. Such an in situ reversible assembly strategy of chemo-photothermal agent also presents a new paradigm for smart and highly efficient disease treatment with reduced side effects.
关键词： chemo-photothermal therapy,doxorubicin,ATP-imaging,AuNP aggregate,intracellular in situ reversible assembly
更新于2025-09-16 10:30:52
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CAN MILD COGNITIVE IMPAIRMENT BE STABILIZED BY SHOWERING BRAIN MITOCHONDRIA WITH LASER PHOTONS?
摘要： There is now substantial evidence that cerebral blood flow (CBF) declines with age. From age 20 to 60, CBF is estimated to dip about 16% and continues to drop at a rate of 0.4%/year. This CBF dip will slowly reduce oxygen/glucose delivery to brain thus lowering ATP energy production needed by brain cells to perform normal activities. Reduced ATP production from mitochondrial loss or damage in the wear-and-tear of aging worsens when vascular risk factors (VRF) to Alzheimer’s disease develop that can accelerate both age-decline CBF and mitochondrial deficiency to a level where mild cognitive impairment (MCI) develops. To date, no pharmacological or any other treatment has been successful in reversing, stabilizing or delaying MCI. For the first time in medical interventions, a non-pharmacological, non-invasive, well-tolerated, easy to perform, free of significant side effects and cost-effective treatment may achieve what virtually all AD treatments in the past have been unable to accomplish. This intervention uses transcranial infrared brain stimulation (TIBS), a form of photobiomodulation (PBM). PBM is a bioenergetic non-ionizing, therapeutic approach using low level light emission from laser or light emitting diodes. PBM has been used in a number of neurological conditions including Parkinson’s disease, depression, traumatic brain injury, and stroke with diverse reported benefits. This brief review examines the impact of reduced energy supply stemming from chronic brain hypoperfusion in the aging brain. In this context, the use of TIBS is planned in a randomized, placebo-controlled study of MCI patients to be done at our University Clinic.
关键词： chronic brain hypoperfusion,laser,ATP,cerebral blood flow,Alzheimer’s disease,mild cognitive impairment,photobiomodulation,axonal transport,neuronal energy,transcranial infrared brain stimulation
更新于2025-09-12 10:27:22
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Multifunctional single-layered graphene quantum dots used for diagnosis of mitochondrial malfunction-related diseases
摘要： Mitochondria are critical organelles in eukaryotes that efficiently generate ATP for various biological activities and any defect in the process of ATP synthesis may lead to mitochondrial dysfunction and directly link to a variety of medical disorders. Monitoring the ATP variations in cells is key for innovative early diagnosis of mitochondrial diseases. Herein, multifunctional single-layered graphene quantum dots (s-GQDs) with bright green emission were constructed, which exhibit strong binding a?nity for ATP and good mitochondria targeting ability. Using the proposed s-GQDs we successfully achieved to discriminate the primary smooth muscle cells isolated from the transgenic mouse (heterozygote Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) 2 C674S knock-in mouse) with mitochondrial disorders or their littermate controls, indicating s-GQDs as promising probes for the study of cell metabolism and mitochondrial malfunction-related diseases, and targeting endoplasmic reticulum stress is an effective way to modulate metabolic pathways relevant to SERCA 2 inactivity mitochondrial dysfunction.
关键词： graphene quantum dots,ATP,mitochondrial disorders,endoplasmic reticulum stress,sarco/endoplasmic reticulum Ca2+ ATPase
更新于2025-09-12 10:27:22