Biosensor for ATP detection via aptamer-modified PDA@POSS nanoparticles synthesized in a microfluidic reactor.

This study introduces aptamer-functionalized polyhedral oligomeric silsesquioxane (POSS) nanoparticles for adenosine triphosphate (ATP) detection where the POSS nanoparticles were synthesized in a one-step, continuous flow microfluidic reactor utilizing thermal polymerization. A microemulsion containing POSS monomers was generated in the microfluidic reactor which was designed to prevent clogging by using a continuous oil flow around the emulsion during thermal polymerization. Surfaces of POSS nanoparticles were biomimetically modified by polydopamine. The aptamer sequence for ATP was successfully attached to POSS nanoparticles. The aptamer-modified POSS nanoparticles were tested for affinity-based biosensor applications using ATP as a model molecule. The nanoparticles were able to capture ATP molecules successfully with an affinity constant of 46.5 [Formula: see text]M. Based on this result, it was shown, for the first time, that microfluidic synthesis of POSS nanoparticles can be utilized in designing aptamer-functionalized nanosystems for biosensor applications. The integration of POSS in biosensing technologies not only exemplifies the versatility and efficacy of these nanoparticles but also marks a significant contribution to the field of biorecognition and sample preparation.

DNA Aptamer Beacon Probe (ABP) for Monitoring of Adenosine Triphosphate Level in SW480 Cancer Cells Treated with Glycolysis Inhibitor 2-Deoxyglucose.

Early cancer screening enables timely detection of carcinogenesis, and aids in prompt clinical intervention. Herein, we report on the development of a simple, sensitive, and rapid fluorometric assay based on the aptamer probe (aptamer beacon probe, ABP) for monitoring the energy-demand biomarker adenosine triphosphate (ATP), an essential energy source that is released into the tumor microenvironment. Its level plays a significant role in risk assessment of malignancies. The operation of the ABP for ATP was examined using solutions of ATP and other nucleotides (UTP, GTP, CTP), followed by monitoring of ATP production in SW480 cancer cells. Then, the effect of a glycolysis inhibitor, 2-deoxyglucose (2-DG), on SW480 cells was investigated. The stability of predominant ABP conformations in the temperature range of 23-91 °C and the effects of temperature on ABP interactions with ATP, UTP, GTP, and CTP were evaluated based on quenching efficiencies (QE) and Stern-Volmer constants (KSV). The optimized temperature for best selectivity of ABP toward ATP was 40 °C (KSV = 1093 M-1, QE = 42%). We have found that the inhibition of glycolysis in SW480 cancer cells by 2-deoxyglucose resulted in lowering of ATP production by 31.7%. Therefore, monitoring and modulation of ATP concentration may aid in future cancer treatment.

A pH-responsive bioassay for sensitive colorimetric detection of adenosine triphosphate based on switchable DNA aptamer and metal ion-urease interactions.

A facile and economic colorimetric strategy was designed for ATP detection by rationally using urease, a pH-responsive molecule, and a metal-mediated switchable DNA probe. By utilizing metal ions as a modulator of urease activity, the concentration of ATP is translated into pH change, which can be readily visualized by naked eye. An unmodified single-stranded DNA probe was designed, which consists of a target binding sequence and two flanked cytosine (C)-rich sequences. This C-rich single-stranded DNA can form a hairpin structure triggered by Ag+ ions via C-Ag+-C base mismatch. Upon introduction of ATP, Ag+-coordinated hairpin DNA structure will be broken and release the included Ag+, thus inhibiting the activity of urease. Conversely, urease can hydrolyze urea and raise pH value of the solution, resulting in the color change of the sensing solution. The proposed assay allows determination of ATP as low as 1.6 nM and shows a satisfactory result in human serum. Because of simple operation and low cost of this method, we believe it has a potential in point-of-care (POC) testing in resource-limited areas. Schematic illustration of pH-responsive colorimetric sensor for ATP detection based on switchable DNA aptamer and metal ion-urease interactions.

One-step and ultrasensitive ATP detection by using positively charged nano-gold@graphene oxide as a versatile nanocomposite.

A versatile nanocomposite was simply prepared based upon the electrostatic adsorption of positively charged gold nanoparticles with negatively charged graphene oxide (nano-gold@GO), and utilized as a novel fluorescence quenching platform for ultrasensitive detection of adenosine triphosphate (ATP). In the designed system, DNA-stabilized Ag nanoclusters (DNA/AgNCs) were used as fluorescent probes, DNA duplex was formed in the presence of ATP, and they can electrostatically adsorb onto the surface of nano-gold@GO to quench the fluorescence signal. Upon the addition of exonuclease III (Exo III), the DNA duplex would be hydrolyzed into DNA fragments and resulted in the recovery of the fluorescence signals due to the diffusion of AgNCs away from nano-gold@GO. Based on these, sensitive detection of ATP was realized with a detection range of 5.0 pM-20 nM. Notably, a good recovery in the range of 94-104% was obtained when detecting ATP in human serum samples, indicating a promising application value in early disease diagnosis. Graphical abstract A functional positively charged nano-gold@graphene oxide was fabricated and utilized as an enhanced fluorescence quenching platform for the detection of ATP, coupled with exonuclease III-assisted signal amplification.

Tunable superamphiphobic surfaces: a platform for naked-eye ATP detection.

A superamphiphobic surface composed of two different size ranges of TiO2 nanoparticles was simply fabricated through spraying the perfluorosilane coated TiO2 nanoparticles suspension dispersing in ethanol. The surface chemistry was finely regulated through gradient UV irradiation-induced organic compound degradation to fabricate surface with gradient solid surface energy or wettability. The fabricated surface shows good droplet sorting ability, which can successfully discriminate ethanol droplets with different concentrations. As a proof-of-concept, the biosensor application of this surface was demonstrated by using it for naked-eye ATP detection. Liquid droplets with different concentrations of ATP after ATP-dependent rolling circle amplification (RCA) can be effectively sorted by the surface. This developed biosensor methodology based on droplet sorting ability of the fabricated surface is energy-efficient and economical which is promising for biosensors, point-of-care testing, and biochemical assays. Graphical abstract ᅟ.

Biomedical applications of nanoflares: Targeted intracellular fluorescence probes.

Nanoflares are intracellular probes consisting of oligonucleotides immobilized on various nanoparticles that can recognize intracellular nucleic acids or other analytes, thus releasing a fluorescent reporter dye. Single-stranded DNA (ssDNA) complementary to mRNA for a target gene is constructed containing a 3'-thiol for binding to gold nanoparticles. The ssDNA "recognition sequence" is prehybridized to a shorter DNA complement containing a fluorescent dye that is quenched. The functionalized gold nanoparticles are easily taken up into cells. When the ssDNA recognizes its complementary target, the fluorescent dye is released inside the cells. Different intracellular targets can be detected by nanoflares, such as mRNAs coding for genes over-expressed in cancer (epithelial-mesenchymal transition, oncogenes, thymidine kinase, telomerase, etc.), intracellular levels of ATP, pH values and inorganic ions can also be measured. Advantages include high transfection efficiency, enzymatic stability, good optical properties, biocompatibility, high selectivity and specificity. Multiplexed assays and FRET-based systems have been designed.

Green fluorescent carbon quantum dots functionalized with polyethyleneimine, and their application to aptamer-based determination of thrombin and ATP.

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. Graphical abstract Green fluorescent carbon quantum dots were functionalized with polyethyleneimine. They were applied to aptamer-based determination of thrombin and ATP. The PEI-functionalized carbon quantum dots (PEI-CDs) have bright green fluorescence are were synthesized by one-step hydrothermal treatment of malic acid and PEI. Employing the PEI-CDs, a fluorometric aptamer-based assay was developed for the determination of thrombin and ATP.

A Label-Free Fluorescent AND Logic Gate Aptasensor for Sensitive ATP Detection.

In this study, a label-free fluorescent, enzyme-free, simple, highly sensitive AND logic gate aptasensor was developed for the detection of adenosine triphosphate (ATP). Double-stranded deoxyribonucleic acid (DNA) with cohesive ends was attached to graphene oxide (GO) to form an aptasensor probe. ATP and single-stranded DNA were used as input signals. Fluorescence intensity of PicoGreen dye was used as an output signal. The biosensor-related performances, including the logic gate construction, reaction time, linearity, sensitivity, and specificity, were investigated and the results showed that an AND logic gate was successfully constructed. The ATP detection range was found to be 20 to 400 nM (R² = 0.9943) with limit of detection (LOD) of 142.6 pM, and the sensitivity range was 1.846 × 106 to 2.988 × 106 M-1. This method for the detection of ATP has the characteristics of being simple, low cost, and highly sensitive.

A Label-Free Fluorescent DNA Calculator Based on Gold Nanoparticles for Sensitive Detection of ATP.

Herein we described a deoxyribonucleic acid (DNA) calculator for sensitive detection of the determination of adenosine triphosphate (ATP) using gold nanoparticles (GNP) and PicoGreen fluorescence dye as signal transducer, and ATP and single-stranded DNA (DNA-M') as activators. The calculator-related performances including linearity, reaction time, logic gate, and selectivity were investigated, respectively. The results revealed that this oligonucleotide sensor was highly sensitive and selective. The detection range was 50⁻500 nmol/L (R² = 0.99391) and the detection limit was 46.5 nmol/L. The AND DNA calculator was successfully used for the ATP detection in human urine. Compared with other methods, this DNA calculator has the characteristics of being label-free, non-enzymic, simple, and highly sensitive.

Label-free and rapid detection of ATP based on structure switching of aptamers.

In this work, an aptamer-based fluorescent strategy for label-free detection of ATP was developed by using Thioflavin T (ThT) as a fluorescence indicator, which can specifically bind with G-quadruplex DNAs to generate enhanced fluorescence intensity. In the absence of ATP, the folded structure of ATP aptamer allows the intercalation of ThT to produce strong fluorescence signal. However, upon ATP binding to the aptamer where ThT intercalated, the conformational change or distortion of the aptamer is large enough to cause much less intercalation of ThT and consequently drastic suppression of the fluorescence intensity. As such, the concentration of ATP could be identified very easily by observing fluorescence changes of this sensing system. This label-free assay could be accomplished very easily and quickly with a "mix-and-detect" detection method and exhibits high sensitivity to ATP with a detection limit of 33 nM in a wide range of 0.1-1000 µM. Furthermore, this proposed method is capable of detecting ATP in human serum and cell extracts. This method offers several advantages such as simplicity, rapidity, low cost, good stability and excellent selectivity, which make it hold great potential for the detection of ATP in bioanalytical and biological studies.

A facile label-free aptasensor for detecting ATP based on fluorescence enhancement of poly(thymine)-templated copper nanoparticles.

A label-free fluorescence assay has been developed for sensitive and selective detection of adenosine triphosphate (ATP) by using poly(thymine) (poly T)-templated copper nanoparticles (CuNPs) as fluorescent indicator. In our design, ATP aptamer was split into two fragments, both of which were elongated with poly T strands that can be utilized as efficient template for the formation of copper nanoparticles through the reduction of copper ions by sodium ascorbate. In the presence of ATP, the two split aptamers could be dragged to form aptamer-ATP aptamer complex, which drew the poly T strands close to each other and induced a remarkable fluorescence enhancement of poly T-templated CuNPs. Thus, an elevated fluorescence enhancement of poly T-templated CuNPs was obtained with the increase in ATP concentration. Under optimized conditions, a good linear range for ATP detection was realized from 100 nM to 100 µM with a detection limit of 10.29 nM. In addition, the application of this biosensing system in complex biological matrix was demonstrated with satisfactory results. This assay provided a simple, label-free, cost-effective, and sensitive platform for the detection of ATP.

Recent advances in fluorescent probes for ATP imaging.

Adenosine-5'-triphosphate (ATP) is a critical biological molecule that functions as the primary energy currency within cells. ATP synthesis occurs in the mitochondria, and variations in its concentration can significantly influence mitochondrial and cellular performance. Prior studies have established a link between ATP levels and a variety of diseases, such as cancer, neurodegenerative conditions, ischemia, and hypoglycemia. Consequently, researchers have developed many fluorescent probes for ATP detection, recognizing the importance of monitoring intracellular ATP levels to understand cellular processes. These probes have been effectively utilized for visualizing ATP in living cells and biological samples. In this comprehensive review, we categorize fluorescent sensors developed in the last five years for ATP detection. We base our classification on fluorophores, structure, multi-response channels, and application. We also evaluate the challenges and potential for advancing new generations of fluorescence imaging probes for monitoring ATP in living cells. We hope this summary motivates researchers to design innovative and effective probes tailored to ATP sensing. We foresee imminent progress in the development of highly sophisticated ATP probes.

PEGylated AIEgens for dual sensing of ATP and H2S and cancer cells photodynamic therapy.

Fluorescent sensors have been widely applied for biosensing, but probes for both multiple analytes sensing and photodynamic therapy (PDT) effect are less reported. In this article, we reported three AIE-based probes anchored with different mass-weight polyethylene glycol (PEG) tails, i.e., TPE-PEG160, TPE-PEG350, and TPE-PEG750, for both adenosine-5'-triphosphate (ATP) and hydrogen sulfide (H2S) detection and also cancer cells photodynamic therapy. TPE-PEGns (n = 160, 350 and 750) contain the tetraphenylethylene-based fluorophore core, the pyridinium and amide anion binding sites, the H2S cleavable disulfide bond, and the hydrophilic PEG chain. They exhibit a good amphiphilic property and can self-assemble nona-aggregation with a moderated red emission in an aqueous solution. Importantly, the size of aggregation, photophysical property, sensing ability and photosensitivity of these amphiphilic probes can be controlled by tuning the PEG chain length. Moreover, the selected probe TPE-PEG160 has been successfully used to detect environmental H2S and image ATP levels in living cells, and TPE-PEG750 has been used for photodynamic therapy of tumor cells under light irradiation.

Development of a Novel ATP Bioluminescence Assay Based on Engineered Probiotic Saccharomyces boulardii Expressing Firefly Luciferase.

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r2 = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

Self-assembled DNA origami-based duplexed aptasensors combined with centrifugal filters for efficient and rechargeable ATP detection.

DNA origami technology has great potential for biosensor applications. Here, we described the construction of a self-assembled DNA origami biosensor for the precise localization of fluorescent aptamers. Due to the molecular weight difference between DNA origami and aptamer, centrifugal filters were used to quantitatively detect adenosine triphosphate (ATP). The ATP-specific aptamer labeled with fluorescence reporter 6-carboxyfluorescein FAM (FAM-aptamer) was selected as the recognition element and signal probe. ATP duplexed aptamers bound to triangular DNA origami by base-complementary pairing, resulting in high fluorescence signals on the origami arrays. The competitive binding of ATP toward the FAM-aptamer triggered the release of FAM-aptamer-ATP complexes from the surface of the origami array, resulting in weakened fluorescence signals. For ATP quantification, 100 kD centrifugal filters were employed, followed by measurement of the fluorescence signal trapped on the origami arrays of the filter device. The successful synthesis of origami-aptamer arrays was characterized by atomic force microscopy, laser confocal microscopy, and electrophoresis. Fluorescence measurements exhibited an excellent linear relationship with logarithms of ATP concentrations within 0.1-100 ng mL-1, with a detection limit of 0.29 ng mL-1. By replacing aptamers and complementary strands, we demonstrated the potential of this method for 17ß-estradiol detection. Considering that the detection mechanism is based on the hybridization and displacement of DNA strands, the detection system had the potential for recharging. Our study provides new insights into applying DNA origami technology in small molecule detection.

Dual-hairpin ligation amplification enabled ultra-sensitive and selective ATP detection for cancer monitor.

Abnormal concentration of ATP is related to many diseases such as Parkinson's disease, hypoglycaemia, inflammation and cancer. However, most of the reported strategies exhibit moderate sensitivity with ∼nM level detection limit and few of them can distinguish ATP from its analogues, such as GTP, CTP, UTP and adenosine. Herein, we report an ultra-sensitive and selective ATP detection strategy that combines dual hairpin ligation-induced isothermal amplification (DHLA) with ATP-dependent enzymatic reaction. A good linear relationship between Cq value and ATP concentration in the range from 16 fM to 160 nM is acquired. Meanwhile, the strategy can distinguish ATP from its analogues with high selectivity. Furthermore, our proposed strategy has been successfully utilized to detect ATP from colon cell line and cell culture media with great potential applications in cell metabolism and cancer diagnosis.

A genetically encoded fluorescent biosensor for monitoring ATP in living cells with heterobifunctional aptamers.

Visualizing the dynamics of ATP in living cells is key to understanding cellular energy metabolism and related diseases. However, the live-cell applications of current methods are still limited due to challenges in biological compatibility and sensitivity to pH. Herein, a novel label-free fluorescent " turn-on " biosensor for monitoring ATP in living bacterias and mammalian cells was developed. This biosensor (Broc-ATP) employed heterobifunctional aptamers to detect ATP with high sensitivity in vitro. In our system, a very useful tandem method was established by combining four Broc-ATPs with 3 × F30 three-way junction scaffold to construct an intracellular biosensor that achieves sufficient fluorescence to respond to intracellular ATP. This intracellular biosensor can be used for sensitive and specific dynamic imaging of ATP in mammalian cells. Hence, this genetically encoded biosensor provides a robust and efficient tool for the detection of intracellular ATP dynamics and 3 × F30 tandem method expands the application of heterobifunctional aptamers in mammalian cells.

Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification.

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 ((+)AuNRs) as an efficient fluorescence quenching platform, coupled with exonuclease Ⅲ (Exo Ⅲ) assisted target recycling amplification. To construct the sensor, DNA template that contained ATP aptamer was used for the formation of Ag nanoclusters 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 Ⅲ, 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 Ⅲ 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.

Fe3O4 NP@ZIF-8/MoS2 QD-based electrochemiluminescence with nanosurface energy transfer strategy for point-of-care determination of ATP.

Herein, Fe3O4 NP@ZIF-8/MoS2 QD-based electrochemiluminescence (ECL) biosensor with nanosurface energy transfer strategy was successfully developed for point-of-care determination of ATP. With the porous structure and poor electron transfer ability, Fe3O4 NP@ZIF-8 complex was first used as an excellent catalyst in ECL. The complex catalyzed the coreactant for more free radicals and hindered the quenching effect of Fe3O4 nanoparticles (NPs) on quantum dots (QDs). In ECL-nanosurface energy transfer (NSET) system, through the specific binding of complementary DNA linked to MoS2 QDs (QDs-cDNA) and aptamer linked to Au NPs, interaction between the point dipole of MoS2 QDs and the collective dipoles of Au NPs quenched ECL signal. When ATP was captured by aptamer, the ECL-NSET system was taken apart, which resulted in the recovery of ECL signal. Moreover, changes of the ECL imaging can be captured by a smartphone, which enabled point-of-care determination of ATP from 0.05 nmol L-1 to 200 nmol L-1 with LOD of 0.015 nmol L-1. With superior specificity and stability, the sensing system showed significant potential about the application of catalysts coated with ZIF and NSET in point-of-care ECL determination.

Highly Selective Fluorescence Sensing and Imaging of ATP Using a Boronic Acid Groups-Bearing Polythiophene Derivate.

A boronic acid groups-bearing polythiophene derivate (L) was designed and synthesized for highly sensitive fluorescence detection of ATP based on a multisite-binding coupled with analyte-induced aggregation strategy. L has a polythiophene backbone as fluorophores and two functional side groups, i.e., quaternary ammonium group and boronic acid group, as multibinding sites for ATP. When various structural analogues such as ADP, AMP, and various inorganic phosphates were added into the aqueous solution of L, only ATP caused a remarkable fluorescence quenching of about 60-fold accompanied by obvious color changes of solution from yellow to purple. The detection limit is estimated to be 2 nM based on 3σ/slope. With the advantage of good water solubility, low toxicity, and highly selective response to ATP, L was successfully utilized as a probe to real-time assay activity of adenylate kinase (ADK) and map fluorescent imaging of ATP in living cells.