Cell Membrane-Anchored AND Logic Gate Aptasensor for Tumor Cell-Specific Imaging with Improved Accuracy.

Accurate and rapid imaging of tumor cells is of vital importance for early cancer diagnosis and intervention. Aptamer-based fluorescence sensors have become a potent instrument for bioimaging, while false positives and on-target off-tumors linked to single-biomarker aptasensors compromise the specificity and sensitivity of cancer imaging. In this paper, we describe a sequential response aptasensor for precise cancer cell identification that is based on a DNA "AND" logic gate. Specifically, the sensor consists of three single-stranded DNA, including the P-strand that can sensitively respond to an acid environment, the L-strand containing the ATP aptamer sequence, and the R-strand for target cell anchoring. These DNA strands hybridize with one another to create a Y-shaped structure (named Y-ALGN). The aptamer in the R-strand is utilized to anchor the sensor to the target cell membrane primarily. Responding to the extracellular acidic environment of the tumor (input 1), the I-motif sequence forms a tetramer structure so that the P-strand is released from the Y-shaped structure and exposes the ATP binding sites in the L-strand. Extracellular ATP, as input 2, continuously operates the DNA aptasensor to complete the logic computation. Upon the sequential response of both protons and ATP molecules, the aptasensor is activated with restored fluorescence on a particular cancer cell membrane. Benefiting from the precise computation capacity of the "AND" logic gate, the Y-ALGN aptasensor can distinguish between MCF-7 cells and normal cells with high accuracy. As a simple and dual-stimuli-responsive strategy, this nanodevice would offer a fresh approach for accurately diagnosing tumor cells.

Framework-Induced Electrochemiluminescence Enhancement of an AIEgen-Based MOF Coupled with Heterostructured TiO2@Ag NPs as an Efficient Coreaction Accelerator for Sensitive Biosensing.

In conventional metal-organic framework (MOF) luminophore-involved electrochemiluminescence (ECL) systems, the aggregation-caused quenching commonly exists for the organic luminescent ligands, limiting the ECL efficiency and detection sensitivity. Herein, by employing the aggregation-induced emission luminogen (AIEgen) 1,1,2,2-tetra(4-carboxylbiphenyl)ethylene (H4TCBPE) as a ligand, one high-efficiency ECL emitter (Zr-MOF) was synthesized through a simple hydrothermal reaction. Compared with H4TCBPE monomers and their aggregates, the resultant Zr-MOF possesses the strongest ECL emission, which is mainly attributed to the framework-induced ECL enhancement. Specifically, the heterostructure was prepared by the deposition of silver nanoparticles on TiO2 microflowers and utilized as an efficient coreaction accelerator. Remarkably, the formative heterojunction can increase the interfacial charge transfer efficiency and promote the carrier separation, facilitating the oxidation of coreactant tripropylamine. In this way, a novel aptamer-mediated ECL sensing platform is constructed, achieving the sensitive analysis of adenosine triphosphate with a low detection limit of 0.17 nM. As a proof-of-concept study, this work may enlighten the rational design of new-type MOF-based ECL materials and expand the application scope of the ECL technology.

Aptamer-Loop DNA Nanoflower Recognition and Multicolor Fluorescent Carbon Quantum Dots Labeling System for Multitarget Living Cell Imaging.

Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

Efficacy of spray flushing in the reprocessing of flexible endoscopes: A randomized controlled trial.

BACKGROUND: Commonly used cleaning brushes in the reprocessing of flexible endoscopes often cause damage within the working channels. AIM: To develop a spray flushing system to achieving effective cleaning of the working channels while minimizing damage. METHODS: This prospective study included 60 used endoscopes and 60 Teflon tubes randomly divided into a control group (n = 30) and an experimental group (n = 30). The material of Teflon tubes was the same as that of the endoscope working channel. Endoscopes in the control group were manually cleaned using traditional cleaning brushes, while those in the experimental group were cleaned using the newly developed spray flushing system. ATP levels, cleanliness, and microbiological testing of the working channels were measured. Additionally, Teflon tubes in the control group underwent 500 passes with a cleaning brush, while those in the experimental group were subjected to the spray flushing system, and channel damage was evaluated. RESULTS: The ATP levels (RLU) in the two groups were 32.5 (13-66) and 26 (16-40), respectively (P > 0.05). Cleanliness scores were 1.5 (1-2) and 1 (1-2), respectively (P > 0.05). Debris was found in 73.3% of the control group, which was significantly higher than 46.7% in the experimental group (P < 0.05). Microbiological tests for both groups yielded negative results. Teflon tube damage in the control group was rated at 4 (4-5.25), which was significantly higher than in the experimental group 4 (3-4) (P < 0.01). CONCLUSION: The spray flushing system demonstrated superior efficacy in removing debris and resulted in less damage to the endoscope working channels compared with traditional cleaning brushes.

Dual-emission red carbon dots for ATP real-time monitoring and quantification to reveal drug and cancer effects on lysosomes.

Monitoring and quantifying ATP levels in vivo is essential to understanding its role as a signaling molecule in tumor progression and therapy. Nevertheless, the real-time monitoring and quantitative assessment of lysosomal ATP remains challenging due to the lack of accurate tools in deep tissues. In this study, based on the crosslinking enhanced emission (CEE) effect, we successfully synthesized red carbon dots (R-CDs) with dual emission properties for efficient quantification of intracellular ATP. The R-CDs emit in the near-infrared range and target lysosomes with rapid detection capabilities, rendering them exceptionally well-suited for directly observing and analyzing the dynamics of lysosomal ATP through live cell imaging techniques. Importantly, R-CDs have proven their efficacy in real-time monitoring of drug stimulus-induced fluctuations in endogenous lysosomal ATP concentration and have also been employed for quantifying and distinguishing lysosomal ATP levels among normal and cancer cell lines. These noteworthy findings emphasize the versatility of the R-CD as a valuable imaging tool for elucidating the functional role of lysosomal ATP in drug screening and cancer diagnostics and hold the promise of becoming a reference tool for deepening our understanding of drug mechanisms of action.

A liquid culture method assisted by ATP analysis for accelerating laboratory experiments on ultraviolet disinfection of airborne bacteria.

The solid culture method for measuring the efficiency of ultraviolet (UV) disinfection of airborne bacteria is time-consuming, typically taking 12-48 h. To expedite such experiments, this study proposed a liquid culture method assisted by adenosine triphosphate (ATP) analysis, as a liquid culture is faster than a solid culture, and measurement of ATP does not require waiting for visible colonies to form. Escherichia coli (E. coli) was used as the experimental bacterium. This study first compared the log reduction of bacteria in liquid as measured by the proposed method and by the traditional solid culture method. The minimum liquid culture time was determined for different bacterial concentration ranges. Finally, the feasibility of the proposed method was validated by UV disinfection experiments on airborne bacteria. The results indicated that the proposed method measured a similar log reduction to that of the solid culture method in liquid experiments. The minimum liquid culture time for E. coli in 105-106 colony forming units (CFU)/mL was 2 h. The validation experiments demonstrated that the proposed method is capable of measuring the UV disinfection efficiency of airborne bacteria. The proposed method can accelerate laboratory experiments on UV disinfection of airborne bacteria, which in turn can support the effective design and utilization of UV disinfection in real life.

Visualizing mitochondrial ATP fluctuations in single cells during photodynamic therapy by In-Situ SERS three-dimensional imaging.

An ultrasensitive strategy for in-situ visual monitoring of ATP in a single living tumor cell during mitochondria-targeted photodynamic therapy (PDT) process with high spatiotemporal resolution was proposed using surface-enhanced Raman scattering (SERS) 3D imaging technique. The nanostructures consisting of Au-Ag2S Janus nanoparticles functionalized with both Au nanoparticles linked by a DNA chain and a mitochondrial-targeting peptide (JMDA NPs) were deliberately employed to target mitochondria. The JMDA NPs exhibit excellent SERS activity and remarkable antitumor activity. The quantization of ATP relies on the intensity of the SERS probes bonded to the DNA, which shows a strong correlation with the generated hot spot between the Janus and the Au. Consequently, spatiotemporally controlled monitoring of ATP in the mitochondria of single living cells during the PDT process was achieved. Additionally, the JMDA NPs demonstrated remarkable capability for mitochondria-targeted PDT, providing significant antitumor effects and superior therapeutic safety both in vitro and in vivo. Our work presents an effective JMDA NPs-based SERS imaging strategy for in-situ and real-time 3D visualization of intracellular ATP in living tumor cells during the mitochondria-targeted PDT process, which enables significant information on the time point of PDT treatment and is beneficial to precious PDT applications in tumor therapy.

Versatile, reusable and highly sensitive SERS-based point-of-care testing microplatform for reliable ATP detection.

The advancement in miniaturized Raman spectrometers, coupled with the single-molecule-level sensitivity and unique fingerprint identification capability of surface-enhanced Raman scattering (SERS), offers great potential for point-of-care testing (POCT). Despite this, accurately quantifying analyte molecules, particularly in complex samples with limited sample volumes, remains difficult. Herein, we present a versatile and reusable SERS microplatform for highly sensitive and reliable quantitative detection of adenosine triphosphate (ATP) in biological fluids. The platform utilizes gold-Prussian blue core-shell nanoparticles modified with polyethyleneimine (Au@PB@PEI NPs), embedded within gold nanoparticle-immobilized capillary-based silica monolithic materials. PB acts as an internal standard, while PEI enhances molecular capture. The periodic, bimodal porous structure of the silica monolithic materials provides uniform and abundant sites for nanoparticle attachment, facilitating rapid liquid permeation, intense SERS enhancement, and efficient enrichment. The platform regulates ATP capture and release through magnesium ions in the liquid phase, eliminating matrix interferences and enabling platform reuse. Integrating efficient molecular enrichment, separation, an interference-free internal standard, a liquid flow channel, and a detection chamber, our platform offers simplicity in operation, exceptional sensitivity and accuracy, and rapid analysis (∼10 min). Employing PB as an internal calibration standard, ratiometric Raman signals (I732/I2123) facilitate precise ATP quantification, achieving a remarkable limit of detection down to 0.62 pM. Furthermore, this platform has been proven to be highly reproducible and validated for ATP quantification in both mouse cerebrospinal fluid and human serum, underscoring its immense potential for POCT applications.

Science behind children's handwashing: action study of 9- to 10-years-old elementary school students in Japan.

Background: Hand washing instructions for children have been implemented in school education to establish good lifestyle habits. However, repeated hand washing through education from early childhood was common for both teachers and children. If this continues, children might assume they already know how to wash their hands, stop taking handwashing instructions seriously, and become increasingly lax about washing their hands. Purpose: This study aimed to develop a new handwashing education method for children. Methods: We applied the adenosine triphosphate (ATP) test to health education on hand washing in elementary schools. This study was conducted as part of a class for elementary school students in October 2023, in Hokkaido, Japan. The subjects were 157 third-grade (9-10 years old) elementary school students. After excluding absent pupils, 147 were included in the analysis. Results: Both pre- and post-education, ATP values after handwashing were lower than those before handwashing. Following the education, children's handwashing behavior improved, with an increase in the number of point washed and appropriate timing of handwashing. Conclusion: The new handwashing education program utilizing the ATP-test succeeded in promoting handwashing behavior among many children. Visualizing handwashing using ATP values was effective in motivating children.

Reconfigurable Optical Sensor for Metal-Ion-Mediated Label-Free Recognition of Different Biomolecular Targets.

Reconfiguration of chemical sensors, intended as the capacity of the sensor to adapt to novel operational scenarios, e.g., new target analytes, is potentially game changing and would enable rapid and cost-effective reaction to dynamic changes occurring at healthcare, environmental, and industrial levels. Yet, it is still a challenge, and rare examples of sensor reconfiguration have been reported to date. Here, we report on a reconfigurable label-free optical sensor leveraging the versatile immobilization of metal ions through a chelating agent on a nanostructured porous silica (PSiO2) optical transducer for the detection of different biomolecules. First, we show the reversible grafting of different metal ions on the PSiO2 surface, namely, Ni2+, Cu2+, Zn2+, and Fe3+, which can mediate the interaction with different biomolecules and be switched under mild conditions. Then, we demonstrate reconfiguration of the sensor at two levels: 1) switching of the metal ions on the PSiO2 surface from Cu2+ to Zn2+ and testing the ability of Cu2+-functionalized and Zn2+-reconfigured devices for the sensing of the dipeptide carnosine (CAR), leveraging the well-known chelating ability of CAR toward divalent metal ions; and 2) reconfiguration of the Cu2+-functionalized PSiO2 sensor for a different target analyte, namely, the nucleotide adenosine triphosphate (ATP), switching Cu2+ with Fe3+ ions to exploit the interaction with ATP through phosphate groups. The Cu2+-functionalized and Zn2+-reconfigured sensors show effective sensing performance in CAR detection, also evaluated in tissue samples from murine brain, and so does the Fe3+-reconfigured sensor toward ATP, thus demonstrating effective reconfiguration of the sensor with the proposed surface chemistry.

See the power in kidney cells with ATP biosensor.

Yamamoto et al. developed an exciting technical advance to examine intracellular adenosine triphosphate levels with single-cell resolution in intact living kidney tissue, including in tubular and vascular segments that lie deep under the kidney surface. The work is a significant advance on prior in vivo biosensor studies, and it allows for mechanistic investigation of alterations in cell metabolism, kidney disease pathobiology, and the effects of drug treatments on energy sources in different kidney cell types.

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.

Biosignature Molecules Accumulate and Persist in Evaporitic Brines: Implications for Planetary Exploration.

The abundance of potentially habitable hypersaline environments in our solar system compels us to understand the impacts of high-salt matrices and brine dynamics on biosignature detection efforts. We identified and quantified organic compounds in brines from South Bay Salt Works (SBSW), where evapoconcentration of ocean water enables exploration of the impact of NaCl- and MgCl2-dominated brines on the detection of potential biosignature molecules. In SBSW, organic biosignature abundance and distribution are likely influenced by evapoconcentration, osmolyte accumulation, and preservation effects. Bioluminescence assays show that adenosine triphosphate (ATP) concentrations are higher in NaCl-rich, low water activity (aw) samples (<0.85) from SBSW. This is consistent with the accumulation and preservation of ATP at low aw as described in past laboratory studies. The water-soluble small organic molecule inventory was determined by using microchip capillary electrophoresis paired with high-resolution mass spectrometry (µCE-HRMS). We analyzed the relative distribution of proteinogenic amino acids with a recently developed quantitative method using CE-separation and laser-induced fluorescence (LIF) detection of amino acids in hypersaline brines. Salinity trends for dissolved free amino acids were consistent with amino acid residue abundance determined from the proteome of the microbial community predicted from metagenomic data. This highlights a tangible connection up and down the "-omics" ladder across changing geochemical conditions. The detection of water-soluble organic compounds, specifically proteinogenic amino acids at high abundance (>7 mM) in concentrated brines, demonstrates that potential organic biomarkers accumulate at hypersaline sites and suggests the possibility of long-term preservation. The detection of such molecules in high abundance when using diverse analytical tools appropriate for spacecraft suggests that life detection within hypersaline environments, such as evaporates on Mars and the surface or subsurface brines of ocean world Europa, is plausible and argues such environments should be a high priority for future exploration. Key Words: Salts-Analytical chemistry-Amino acids-Biosignatures-Capillary electrophoresis-Preservation. Astrobiology 24, 795-812.

A naphthyl appended ninhydrin based colorimetric chemosensor for Cu2+ ion: Detection of cysteine and ATP.

A ninhydrin-based colorimetric chemosensor (LH) was synthesized using 3-hydroxy-2-naphthoic hydrazide and 11H-indeno[1,2-b]quinoxalin-11-one. It was characterized by spectroscopic and single crystal X-ray diffraction techniques. In a semi-aqueous (MeOH/HEPES) system, LH displayed a characteristic chromogenic change from colorless to yellow upon adding Cu2+ ion, with the appearance of a new peak at λmax = 460 nm. A 1:1 binding stoichiometry between LH and Cu2+ ion has been found, with LOD = 2.3 µM (145 ppb) and LOQ = 8 µM (504 ppb). Based on experimental results the formula of [Cu(L)Cl(H2O)2] (1) was assigned and this in-situ generated 1 was found to exhibit a discoloration of upon gradual addition of cysteine (LOD = 60 nM) as well as ATP (LOD = 130 nM) having 1:2 and 1:1 stoichiometry respectively. The LH was useful for recognition of Cu2+ ion in real water samples and on filter paper strips. A two-input-two-output logic gate circuitry was also constructed by employing 1 and cysteine. The DFT/TDDFT calculations performed on LH and 1 were consistent with experimental findings. The binding affinity of LH towards HSA and BSA were determined with HSA having greater affinity than BSA, which was also supported by theoretical calculations.

Enhancing Kinase Activity Detection with a Programmable Lanthanide Metal-Organic Framework via ATP-to-ADP Conversion.

Precise modulation of host-guest interactions between programmable Ln-MOFs (lanthanide metal-organic frameworks) and phosphate analytes holds immense promise for enabling novel functionalities in biosensing. However, the intricate relationship between these functionalities and structures remains largely elusive. Understanding this correlation is crucial for advancing the rational design of fluorescent biosensor technology. Presently, there exists a large research gap concerning the utilization of Ln-MOFsto monitor the conversion of ATP to ADP, which poses a limitation for kinase detection. In this work, we delve into the potential of Ln-MOFs to amplify the fluorescence response during the kinase-mediated ATP-to-ADP conversion. Six Eu-MOFs were synthesized and Eu-TPTC ([1,1':4',1″]-terphenyl-3,3'',5,5''-tetracarboxylic acid) was selected as a ratiometric fluorescent probe, which is most suitable for high-precision detection of creatine kinase activity through the differential response from ATP to ADP. The molecular -level mechanism was confirmed by density functional theory. Furthermore, a simple paper chip-based platform was constructed to realize the fast (20 min) and sensitive (limit of detection is 0.34 U/L) creatine kinase activity detection in biological samples. Ln-MOF-phosphate interactions offer promising avenues for kinase activity assays and hold the potential for precise customization of analytical chemistry.

Synthesis of a metal-organic framework Cu-Mi-UiO-66-based fluorescent nanoprobe for the simultaneous sensing and intracellular imaging of GSH and ATP.

This study reports a fluorescent nanoprobe operated in fluorescence turn-on mode for simultaneously sensing and imaging intracellular GSH and ATP. By using maleimide-derivatives as the ligand, the bimetallic nanoscale metal-organic framework (NMOF) Cu-Mi-UiO-66 has been synthesized for the first time using a straightforward one-step solvothermal approach, serving as a GSH recognition moiety. Subsequently, a Cy5-labeled ATP aptamer was assembled onto Cu-Mi-UiO-66 via strong coordination between phosphate and zirconium, π-π stacking and electrostatic adsorption to develop the dual-responsive fluorescence nanoprobe Cu-Mi-UiO-66/aptamer. Due to the photoinduced electron transfer (PET) effect between maleimide groups and the benzene ring of the ligand and the charge transfer between Cy5 and the Zr(IV)/Cu(II) bimetal center of the NMOF, the Cu-Mi-UiO-66/aptamer exhibits a fluorescence turn-off status. The Michael addition reaction between the thiol group of GSH and the maleimide on the NMOF skeleton results in turning on of the blue fluorescence of Cu-Mi-UiO-66. Meanwhile, upon specific interaction with ATP, the aptamer changes into internal loop structures and detaches from Cu-Mi-UiO-66, resulting in turning on of the red fluorescence of Cy5. The nanoprobe demonstrated an excellent sensing performance with a good linear range (GSH, 5.0-450.0 µM; ATP, 1.0-50.0 µM) and a low detection limit (GSH, 2.17 µM; ATP, 0.635 µM). More importantly, the Cu-Mi-UiO-66/aptamer exhibits good performance for tracing intracellular concentration variations of GSH and ATP in living HepG2 cells under different stimulations. This study highlights the potential of NMOFs for multiplexed analysis and provides a valuable tool for tumor microenvironment research and early cancer diagnosis.

Aptamer responsive DNA Functionalized hydrogels electrochemiluminescence biosensor for the detection of adenosine triphosphate.

DNA hydrogel represents a noteworthy biomaterial. The preparation of biosensors by combining DNA hydrogel with electrochemiluminescence can simplify the modification process and raise the experimental efficiency. In this study, an electrochemiluminescence (ECL) biosensor based on DNA hydrogel was fabricated to detect adenosine triphosphate (ATP) simply and quickly. CdTe-Ru@SiO2 nanospheres capable of ECL resonance energy transfer (RET) were synthesized and encapsulated CdTe-Ru@SiO2 in the DNA hydrogel to provide strong and stable ECL signals. DNA hydrogel avoided the labeling of ECL signal molecules. The aptamer of ATP as the linker of the hydrogel for the specificity of ATP detection. The cross-linked structure of the aptamer and the polymer chains was opened by ATP, and then the decomposition of the DNA hydrogel initiated the escape of CdTe-Ru@SiO2 to generate an ECL signal. The designed biosensor detected ATP without too much modification and complex experimental steps on the electrode surface, with good specificity and stability, and a wide linear range. The detection range was 10-5000 nM, and the detection limit was 6.68 nM (S/N = 3). The combination of DNA hydrogel and ECL biosensor provided a new way for clinical detection of ATP and other biomolecule.

Miniaturizable Chemiluminescence System for ATP Detection in Water.

We present the design, fabrication, and testing of a low-cost, miniaturized detection system that utilizes chemiluminescence to measure the presence of adenosine triphosphate (ATP), the energy unit in biological systems, in water samples. The ATP-luciferin chemiluminescent solution was faced to a silicon photomultiplier (SiPM) for highly sensitive real-time detection. This system can detect ATP concentrations as low as 0.2 nM, with a sensitivity of 79.5 A/M. Additionally, it offers rapid response times and can measure the characteristic time required for reactant diffusion and mixing within the reaction volume, determined to be 0.3 ± 0.1 s. This corresponds to a diffusion velocity of approximately 44 ± 14 mm2/s.

Sensitive aptasensing of ATP based on a PAM site-regulated CRISPR/Cas12a activation.

The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.

CRISPR/Cas12a trans-cleavage mediated photoelectrochemical biosensor based on zeolitic imidazolate framework-67 for ATP determination.

An efficient PEC biosensor is proposed for ATP detection based on exciton energy transfer from CdTe quantum dots (CdTe QDs) to Au nanoparticles (AuNPs), integrating CRISPR/Cas12a trans-cleavage activity and specific recognition of ZIF-67 to ATP. Exciton energy transfer between CdTe QDs and AuNPs system is firstly constructed as photoelectrochemical (PEC) sensing substrate. Then, the activator DNAs, used to activate CRISPR/Cas12a, are absorbed on the surface of ZIF-67. In the presence of ATP, the activator DNAs are released due to more efficient adsorption of ZIF-67 to ATP. The released activator DNA activates trans-cleavage activity of CRISPR/Cas12a to degrade ssDNA on the electrode, leading to the recovery of photocurrent due to the interrupted energy transfer. Benefiting from the specific recognition of ZIF-67 to ATP and CRISPR/Cas12a-modulated amplification strategy, the sensor is endowed with excellent specificity and high sensitivity.