ATP Aptamer Research Articles

Reducing Measurement Deviation by Metastable DNA Probes for Aptamer Thermodynamic Characterization.

DNA reaction equilibrium-based calculations have great potential in thermodynamic characterization, but their widespread applications are hindered by significant measurement deviation of equilibrium concentration. Here, we report the advantages of metastable DNA hybridization in reducing quantification deviation of equilibrium concentration and propose a universal and standardized strategy for measuring aptamer binding energy, termed metastable DNA reference calorimetry (MDRC). We built different MDRC-based algorithms tailored to different aptamer binding models, enabling the calculation of thermodynamic parameters for aptamers with one or more binding sites. Our correlative model, considering the cross-effects between different binding sites, showed that for ATP aptamers with two binding sites, binding of the first ATP molecule would decrease its affinity for the second at low temperatures and even completely inhibit this binding at high temperatures. Moreover, the thermodynamic parameters of protein-specific aptamers were calculated to elucidate the universality of the method. The successful analysis of cell-specific aptamers further demonstrated MDRC's applicability in complex biological systems.

An Entropy-Driven Multipedal DNA Walker Microsensor for In Situ Electrochemical Detection of ATP.

Microelectrode- and nanoelectrode-based electrochemistry has become a powerful tool for the in situ monitoring of various biomolecules in vivo. However, two challenges limit the application of micro- and nanoelectrodes: the difficulty of highly sensitive detection of nonelectroactive molecules and the specific detection of target molecules in complex biological environments. Herein, we propose an electrochemical microsensor based on an entropy-driven multipedal DNA walker for the highly sensitive and selective detection of ATP. An ATP aptamer was immobilized on the microelectrode surface to form the DNA walker track for selective ATP recognition. Multiple walking "legs" were attached to quantum dots to create the multipedal DNA walker. ATP was selectively captured by the ATP aptamer, triggering the autonomous movement of the multipedal DNA walker on the microelectrode interface, which brought methylene blue (MB) closer to the microelectrode interface, realizing a cascade signal amplification. Additionally, ATP release induced by hypotonic conditions in BV2 cells was successfully monitored, indicating that increased cell volume enhances ATP release. This multipedal DNA walker microsensor offers a novel strategy for in situ, highly sensitive, and selective monitoring of nonelectroactive biomolecules in vivo, which is crucial for investigating the neurochemical processes involved in neurological diseases.

A hydrogel film microarray stabilizes very short structure switching aptamer duplexes to achieve enhanced sensing of small molecules

The reduction of affinity associated with the modifications required to create FRET- aptamer constructs represents a key challenge hindering the practical adaptation of such systems for small molecule detection. While the use of shorter and rationally positioned quencher stems is desirable to enhance target-induced fluorescence recovery, the weak hybridization potential of such stems results in high levels of background fluorescence and poor sensitivity. Herein, we introduce a hydrogel microarray sensor able to thermally stabilize very short aptamer duplexes (i.e., quencher stems ≤10-bp), resulting in improved FRET efficiency and reduced background levels across a broad range of conditions. The optimal hydrogel microarray can physically entrap >75 % of loaded aptamer reporters while maintaining high accessibility to the target molecules, enabling accurate quantification of dose-responsive affinity interactions. Using an ATP aptamer reporter, the hydrogel sensor can achieve a binding affinity as low as 14.7 µm − comparable to the native aptamer – and very low limits of detection (LOD) of 1.2 µm in a pure buffer and 4.6 µm in 50 % serum. Moreover, the developed hydrogel microarray is reusable, amenable to printing fabrication, and provides protection against nuclease-based degradation. This simple approach thus holds promise for advancing microarray aptamer technology in critical biosensing applications.

Engineering Logic‐Gated i‐Motif/G‐Quadruplex (iG4) Hybrid Structures for DNA Nanotweezer‐Based Aptasensing

AbstractGiven high levels of both K+ and H+ in the tumor microenvironment, G‐quadruplex (G4) and i‐motif can in principle be utilized as two cooperative modules to build logic‐gated DNA nanodevices for microenvironment recognition and targeting. Combined use of G4 and i‐motif in DNA nanoassemblies, however, usually causes the uncontrolled DNA aggregation. To address this trouble, we well design a novel i‐motif/G‐quadruplex (iG4) hybrid structure that integrates two four‐stranded DNA helices in a folding topology, with a parallel mini‐duplex flanking at the 5’ end to provide a binding site for fluorescent ligands. This design enables that the folded and lighting‐up DNA structure is favored by H+ and K+ together, consistent with a two‐input AND gate behavior. We then employ the iG4 structure to guide a DNA nanotweezer that is clamped by an altered split ATP aptamer, which brings a proximity effect contributing to the proper folding of iG4 in slightly acidic microenvironments and enables the sensitive detection of endogenous ATP in cancer cell lysates.

A Fluorescence Enhancement Sensor Based on Silver Nanoclusters Protected by Rich-G-DNA for ATP Detection.

In this study, a turn-on fluorescence sensor for the detection of adenosine 5'-triphosphate (ATP) was developed and tested using ATP-DNA2-Ag NCs. The results showed that the fluorescence of ATP-DNA2-Ag NCs was significantly enhanced with the addition of ATP. The fluorescence enhancement was a result of the specific binding activity of the ATP aptamer and ATP, which caused G-rich sequences to approach the dark DNA-Ag NCs, owing to the alteration in ATP aptamer conformation. The proposed sensor demonstrated a good linear range of 18-42 mM and a limit of detection (LOD) of 2.8 μM. The sensor's features include sensitivity, selectivity, and simple operation. In addition, the proposed sensor successfully measured ATP in 100-fold diluted fetal bovine serum.

Enzyme-responsive aptasensing based on the ATP aptamer and benzothiazole-coumarin G-quadruplex fluorescent probe for label-free detection of adenosine deaminase activity and its inhibitor

Adenosine deaminase (ADA) is a vital enzyme for regulating the biological process in humans and its abnormal expression level is related to various severe diseases. Nevertheless, conventional approaches for detecting ADA activity are hindered by the drawbacks of complexity and limited detection performances. To address these challenges, we here designed an ADA-responsive aptasensing strategy based on a synthesized benzothiazole-coumarin G-quadruplex (G4) fluorescent probe, which can bind to the G-rich ATP aptamer (G-APA) and form the high-contrast fluorescent complex (S1/G-APA). While the fluorescence of S1/G-APA could be quenched by ATP competition due to the allosteric process, the deamination in ATP catalyzed by ADA would recover the fluorescence of S1/G-APA and allow a facile, turn-on, label-free, and selective detection of ADA activity. This proposed method exhibits favorable detection performances for ADA activity analysis with a wide detection linear range varied from 1 to 100 U/L and a detection limit of 0.82 U/L. Meanwhile, it can be applied for ADA inhibitor (DCF) evaluation and provide a turn-off and sensitive detection performances. Inspired by these detection efficacies, the serum samples spiked with ADA and DCF have been investigated to present the satisfied recovery rates and detection reliability. Collectively, this method will provide a promising way for portable and efficient ADA activity quantification and evaluating the inhibition efficiencies of ADA inhibitors, which hold the potential in promoting ADA-related sensing and pharmaceutical research.

PH-dependent binding of ATP aptamer to the target and competition strands: Fluorescent melting curve fitting study

The pH varies in different tissues and organelles and also changes during some diseases. In this regard, the application of molecular switches that use a competition-based aptamer switch design in biological systems requires studying the thermodynamics of such systems at different pH values. In this work, we studied the binding of the classical ATP aptamer to ATP and competition strands under different pH and ionic conditions using fluorescent melting curve analysis. We have developed an original approach to processing source data from a PCR thermal cycler. It is based on constructing a thermodynamic model of the melting profile and the subsequent fit of experimental curves within this model. We have shown that this approach enables us to narrow the temperature region under study to the width of the melting region without a significant loss in the quality of the result. This impressively expands the application area of this approach compared to frequently used techniques that require mandatory measurement of the signal outside the melting region. The results obtained by the method showed that the thermodynamic parameters of the ATP aptamer and its duplexes with competition strands change depending on pH. Therefore, molecular switches that use a competition strand to the ATP aptamer may have a pH-dependent sensitivity that has not been previously considered. This should be taken into account for future rational design of similar systems.

DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels.

Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for splitGFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator splitGFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated splitGFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence.

Passing Behavior of Oligonucleotides through a Stacked DNA Nanochannel with Featured Path Design.

DNA nanotechnology has emerged as a useful tool for constructing artificial channels penetrating the lipid bilayer. In this work, we introduce a stacked DNA origami nanochannel device characterized by a width-variable pathway, consisting of narrow entrance and exit channels coupled with a wide, modifiable lumen. This design modulates the translocation behavior of oligonucleotides, revealing distinct stages of signal patterns in the recorded current traces. The observed prolonged dwell times indicate oligonucleotide retention, specifically due to the transition from the wide lumen to the narrower exit channel, while variations in current recovery between events suggested intermediate channel states between conducting and blocking. Further, by incorporating sequence-specific overhangs within the channel lumen, we achieved unique asymmetric current profiles during ATP aptamer translocation events. Featured stages also highlighted the aptamer binding dynamics and ATP-induced release. The distinguished oligonucleotide passing behaviors afforded by the stacked DNA origami channel with interior decoration demonstrated the strategic and profitable attempts at DNA nanochannel engineering for nanodevice development and applications.

Construction of an exogenously and endogenously Co-activated DNA logic amplifier for highly reliable intracellular MicroRNA imaging

DNA-based molecular amplifiers offer significant promise for molecular-level disease diagnosis and treatment, yet tailoring their activation for precise timing and localization remains a challenge. Herein, we’ve pioneered a dual activation strategy harnessing external light and internal ATP to create a highly controlled DNA logic amplifier (FDLA) for accurate miRNA monitoring in cancer cells. The FDLA was constructed by tethered the two functionalized catalytic hairpin assembly (CHA) hairpin modules (ATP aptamer sealed hairpin aH1 and photocleavable (PC-linker) sites modified hairpin pH2) to DNA tetrahedron (DTN). The FDLA system incorporates ATP aptamers and PC-linkers as logic control units, allowing them to respond to both exogenous UV light and endogenous ATP present within cancer cells. This response triggers the release of CHA hairpin modules, enabling amplified FRET miRNA imaging through an AND-AND gate. The DTN structure could improve the stability of FDLA and accelerate the kinetics of the strand displacement reaction. It is noteworthy that the UV and ATP co-gated DNA circuit can control the DNA bio-computing at specific time and location, offering spatial and temporal capabilities that can be harnessed for miRNA imaging. Furthermore, the miRNA-sensing FDLA amplifier demonstrates reliable imaging of intracellular miRNA with minimal background noise and false-positive signals. This highlights the feasibility of utilizing both exogenous and endogenous regulatory strategies to achieve spatial and temporal control of DNA molecular circuits within living cancer cells. Such advancements hold immense potential for unraveling the correlation between miRNA and associated diseases.

Electrochemiluminescent quenching of luminol–doped Zr–MOFs for ATP detection through scavenging ROS using gallic acid–capped Au nanoparticles

In the present study, the highly efficient electrochemiluminescence (ECL) luminophores of Zr-based metal–organic frameworks (MOFs) coordinating with the amino group of luminol (Zr–Lu–MOFs) were developed for fabricating a sensing platform. The ECL emission of a Zr–Lu–MOF/H2O2 system with strong and stable ECL signals was triggered through cyclic voltammetry. Adenosine triphosphate (ATP) was firmly assembled on Zr–Lu–MOF because of the high affinity of Zr4+ toward phosphate groups. ATP was detected using gallic acid (GA)-capped Au (GA@Au) nanoparticles combined with an ATP aptamer. The GA@Au nanoparticles quenched the ECL behavior of the luminol/H2O2 system, and the quenching mechanism was ascribed to the GA scavenging superoxide radical (O2•−) that was generated via the electron redox reaction in H2O2. Under optimal experiments, an ECL biosensor with a linear range of 0.100–1000 nM and a detection limit of 0.0353 nM (S/N = 3) was designed for the selective and sensitive detection of ATP. Thus, the proposed ECL biosensor exhibited good performance with high stability and acceptable fabrication reproducibility, providing a valuable strategy for clinical diagnostics and therapeutics.

Electrochemical Monitoring of Sphingosine-1-phosphate-Induced ATP Release Using a Microsensor Based on an Entropy-Driven Bipedal DNA Walker.

Neuropathic pain is a chronic and severe syndrome for which effective therapy is insufficient and the release of ATP from microglia induced by sphingosine-1-phosphate (S1P) plays a vital role in neuropathic pain. Therefore, there is an urgent demand to develop highly sensitive and selective ATP biosensors for quantitative monitoring of low-concentration ATP in the complex nervous system, which helps in understanding the mechanism involved in neuropathic pain. Herein, we developed an electrochemical microsensor based on an entropy-driven bipedal DNA walker. First, the microsensor specifically recognized ATP via ATP aptamers, initiating the entropy-driven bipedal DNA walker. Subsequently, the bipedal DNA walker autonomously traversed the microelectrode interface, introducing methylene blue to the electrode surface and achieving cascade signal amplification. This microsensor showed excellent selectivity, stability, and a low limit of detection at 1.13 nM. The S1P-induced ATP release from BV2 cells was successfully monitored, and it was observed that dicumarol could inhibit this release, suggesting dicumarol as a potential treatment for neuropathic pain. The microsensor's small size exhibited significant potential for monitoring ATP level changes in neuropathic pain in vivo, which provides a new strategy for in situ and quantitative monitoring of nonelectroactive biomolecules associated with neurological diseases.

G-Quadruplex Structure in the ATP-Binding DNA Aptamer Strongly Modulates Ligand Binding Activity.

Secondary structures formed by single-stranded DNA aptamers can allow for the binding of small-molecule ligands. Some of these secondary structures are highly stable in solution and are great candidates for use in the development of molecular tools for biomarker detection, environmental monitoring, and others. In this paper, we explored adenosine triphosphate (ATP)-binding aptamers for the simultaneous detection of two small-molecule ligands: adenosine triphosphate (ATP) and thioflavin T (ThT). The aptamer can form a G-quadruplex (G4) structure with two G-quartets, and our results show that each of these quartets is equally involved in binding. Using fluorescently labeled and label-free methods, we further explored the role of the G4 motif in modulating the ligand binding property of the aptamer by making two extended variants that can form three or four G-quartet G4 structures. Through equilibrium binding and electrospray ionization mass spectrometry (ESI-MS) analysis, we observed a stronger affinity of aptamers to ATP by the variant G4 constructs relative to the native aptamer (Kd range of 0.040-0.042 μM for variants as compared to 0.15 μM for the native ATP aptamer). Additionally, we observed a dual binding of ThT and ATP to the G4 constructs in the label-free and ESI-MS analyses. These findings together suggest that the G4 motif in the ATP aptamer is a critical structural element that is required for optimum ATP binding and can be modulated for the binding of multiple ligands. These findings are instrumental for designing smart molecular tools for a wide range of applications, including biomarker monitoring and ligand binding studies.

An electrochemiluminescence microsensor based on DNA-silver nanoclusters amplification for detecting cellular adenosine triphosphate.

Adenosine triphosphate (ATP), as the primary energy source, plays vital roles in many cellular events. Developing an efficient assay is crucial to rapidly evaluate the level of cellular ATP. A portable and integrated electrochemiluminescence (ECL) microsensor array based on a closed bipolar electrode (BPE) was presented. In the BPE unit, the ECL chemicals and oxidation/reduction were separated from the sensing chamber. The ATP aptamer was assembled with single-stranded DNA (ssDNA) in the sensing chamber. ATP capture made the aptamer disassemble from the ssDNA and facilitated DNA-templated silver nanocluster (Ag NC) generation by the target-rolling circle amplification (RCA) reaction. The guanine-rich padlock sequence produced tandem periodic cytosine-rich sequences by the RCA, inducing Ag NC generation in the cytosine-rich region of the produced DNA strands through Ag+ reduction. The in situ Ag NC generation enhanced the circuit conductivity of the BPE and promoted the ECL reaction of [Ru(bpy)2dppz]2+/tripropylamine in the anodic reservoir. On this ECL microsensor, a good linear relationship of ATP was achieved ranging from 30 to 1000 nM. The ATP content in HepG2 cells was selectively and sensitively determined without complex pretreatment. The ATP amount of 25 cells could be successfully detected when a sub-microliter sample was loaded.

Turn-on fluorescent nanoprobe for ATP detection based on DNA-templated silver nanoclusters.

A turn-on fluorescence nanoprobe was constructed for the determination of adenosine 5'-triphosphate (ATP) based on DNA-templated silver nanoclusters (DNA-AgNCs). The significant enhancement fluorescence intensity of DNA-AgNCs in the presence of ATP is due to the high special binding affinity between ATP and the aptamer, resulting in the environment of DNA-AgNCs with darkish fluorescence lying at one terminus of DNA slightly altering owing to the change of ATP aptamer conformation. A good linear range runs from 9 to 24 mM with a satisfactory detection limit of 3 μM. Furthermore, the proposed nanoprobe exhibited good performance for ATP detection in diluted fetal bovine serum.

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.

Label-free chemiluminescent CRISPR/Cas12a biosensing strategy for the detection of nucleic acids and non-nucleic acids utilizing DNAzyme Supernova

CRISPR/Cas12a-based biosensors typically output fluorescent, colorimetric, and electrochemical signals, relying on reporters that suffer from low sensitivity, high cost, and low stability. Herein, we introduced a label-free chemiluminescent (CL) reporting system based on a DNAzyme known as “Supernova”. Supernova catalyzes the translocation of phosphate from a 1,2-dioxetane substrate (specifically, CDP-Star) to its 5′-OH ends, thereby triggering a CL reaction that produces light. Upon activation by the target, Cas12a cleaves Supernova, rendering it inactive in reacting with CDP-Star. Without pre-amplification, this biosensor can detect synthetic DNA corresponding to the SARS-CoV-2 N gene and monkeypox virus (MPXV) F3L gene with detection limits (LOD) of 1.5 and 2.4 pM, respectively. Integration with pre-amplification further enables the detection of SARS-CoV-2 and MPXV genes down to 1 copy/μL in real samples. Furthermore, the application of this CL system was expanded to the detection of non-nucleic acids with the integration of ATP aptamers, resulting in improved sensitivity in ATP biosensing compared to fluorescent assays. Overall, this work represents the first development of a label-free CL Cas12a reporting system, demonstrating its enhanced performance and broad applicability in synthetic biology-enabled sensing technology.

Nucleic acid-MOF nanoparticle conjugates for NIR/ATP-driven synergetic photo-chemotherapy with hypoxia relief

Herein, a specific kind of nano-platform (ICG-PFH/MOF/DNA-Dox) was developed not only for targeted fluorescence imaging-guided photo-chemotherapy, but also for modulation of hypoxic tumor microenvironment for cancer treatment. Such platform was prepared with metal–organic framework (MOF) as core to serve as matrices for storage of indocyanine green (ICG) and perfluorohexane (PFH), and with double-strand nucleic acids (dsDNA) as shell, which is encoded with both ATP aptamer and AS1411 aptamer as well as used for doxorubicin (Dox) anchoring. Thereout, ICG-PFH/MOF/DNA-Dox reveals active targeting to tumor by recognizing nucleolin receptor sites in cancer cell membranes, following controlled release of Dox in mitochondria due to ATP-triggered dsDNA dissociation to achieve chemotherapy (CT). Under a single laser irradiation, ICG can serve both as photothermal agent to emit heat for photothermal therapy (PTT), and as photosensitizer to generate reactive oxygen species for photodynamic therapy (PDT) of tumors. The loaded PFH acts as O2 self-supplying agent for hypoxia-relieved PDT and CT. Interestingly, the heat from PTT can also help to melt dsDNA to accelerate the Dox release, enhancing CT efficiency. To this end, ICG-PFH/MOF/DNA-Dox shows surprising effect in inhibiting tumor growth both in vitro and in vivo via hypoxia-relieved synergistic PTT/PDT/CT.

A spatially-controlled DNA triangular prism nanomachine for AND-gated intracellular imaging of ATP in acidic microenvironment.

ADNA triangular prism nanomachine (TPN)-based logic device for intracellular AND-gated imaging of adenosine triphosphate (ATP)has been constructed. By using i-motif sequences and ATP-binding aptamers as logic control units, the TPN logic device is qualified to respond to the acidic environment and ATP in cancer cell lysosomes. Once internalized into the lysosome, the specific acidic microenvironment in lysosome causes the i-motif sequence to fold into a tetramer, resulting in compression of DNA tri-prism. Subsequently, the split ATP aptamer located at the tip of the collapsed triangular prism binds stably to ATP, which results in the fluorescent dyes (Cy3 and Cy5) modified at the ends of the split aptamer being in close proximity to each other, allowing Förster Resonance Energy Transfer (FRET) to occur. The FRET signals are excited at a wavelength of 543 nm and can be collected within the emission range of 646-730 nm. This enables the precise imaging of ATP within a cell. We also dynamically operate AND logic gates in living cells by modulating intracellular pH and ATP levels with the help of external drugs. Owing to the AND logic unit on TPN itcan simultaneously recognize two targets and give corresponding intelligent logic judgment via imaging signal output. Theaccuracy of molecular diagnosis of cancer can be improved thuseliminating the false positive signal of single target-based detection. Hence, this space-controlled TPN-based logical sensing platform greatly avoids sensitivity to extracellular targets during the cell entry process, providing a useful tool for high-precision imaging of the cancer cell's endogenous target ATP.

Small-Molecule Analysis Based on DNA Strand Displacement Using a Bacteriorhodopsin Photoelectric Transducer: Taking ATP as an Example.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.