Specific Aptamer Research Articles

Targeted Enrichment of Nucleic Acid Bionic Arms Enhances the Hydrolysis Activity of Nanozymes for Degradation and Real-Time Monitoring of Organophosphorus Pesticides in Water.

Organophosphorus pesticides (OPs) pose significant environmental and health risks, and their detoxification through catalytic hydrolysis using zirconium-based metal-organic frameworks (Zr-MOFs) has attracted considerable interest due to the strong Lewis acid metal ions. Albeit important, the defects of the materials for OP hydrolysis (e.g., poor degradation efficiency, rate, and selectivity) limit their further application. Herein, a nucleic acid bionic arm-modified biomimetic nanozyme (MOF-808-Apt) was designed through a Zr-MOF and a specific aptamer against OPs, which was employed for the efficient and selective degradation of OPs. At the system, the functionalized biomimetic nanozyme can continuously capture trace OPs onto its catalytic sites for degradation with the fabricated nucleic acid bionic arms, significantly improving their catalytic activities compared to bare MOF-808 using paraoxon as a model of OPs, providing better performances including (i) an excellent degradation efficiency, boosting from 4 to over 60% within 6 min; (ii) a satisfactory catalytic rate (the pseudo-first-order rate constants of paraoxon hydrolysis improved from 0.09 to 0.14 min-1); and (iii) good selective degradation because of aptamers used. Besides, this dynamic degradation process could be visually recorded in real time with high sensitivity (limit of detection, 0.18 μM) because of the obvious color change of the reaction solution and signal amplification ascribed to increasing local concentrations of targets by the nucleic acid bionic arms. Summarily, this work provides a new strategy for the effective and selective degradation of typical OPs and concurrent monitoring of their dynamic degradation process.

Serum assisted PD-L1 aptamer screening for improving its stability

Aptamers have shown potential for diagnosing clinical markers and targeted treatment of diseases. However, their limited stability and short half-life hinder their broader applications. Here, a real sample assisted capture-SELEX strategy is proposed to enhance the aptamer stability, using the selection of specific aptamer towards PD-L1 as an example. Through this developed selection strategy, the aptamer Apt-S1 with higher binding affinity and specificity towards PD-L1 was obtained as compared to the aptamer Apt-A2 which was screened by the traditional capture-SELEX strategy. Moreover, Apt-S1 exhibited a greater PD-L1 binding associated conformational change than Apt-A2, indicating its suitability as a biorecognition element. These findings highlight the potential of Apt-S1 in clinical applications requiring robust and specific targeting of PD-L1. Significantly, Apt-S1 exhibited a lower degradation rate in 10% diluted serum or pure human serum, under the physiological temperature and pH value, compared to Apt-A2. This observation suggested that Apt-S1 possesses higher stability and is more resistant to damage caused by the serum environmental factors, highlighting the superior stability of Apt-S1 over Apt-A2. Furthermore, defatted and deproteinized serum were used to investigate the potential reasons for the improved stability of Apt-S1. The results hinted that the pre-adaptation to nucleases present in serum during the selection process might have contributed to its higher stability. With its improved stability, higher affinity and specificity, Apt-S1 holds great potential for applications in PD-L1 assisted cancer diagnosis and treatment. Meanwhile, the results obtained in this work provide further evidence of the advantages of the real capture-SELEX strategy in improving aptamer stability compared to the traditional strategy.

Microfluidic-SERS platform with in-situ nanoparticle synthesis for rapid E. coli detection in food.

A microfluidic-surface enhanced Raman spectroscopy (SERS) platform for rapid detection of Escherichia coli in food products is proposed. By implementing a Y-junction serpentine microfluidic channel, we achieved in-situ synthesis of silver nanoparticles (AgNPs), for enhancing SERS signal intensity. The synthesis of AgNPs was guided by specific aptamers bound to the bacterial cell, which facilitated formation of nanoparticles. This aptamer guided in-situ synthesis ensured specificity and accuracy in detecting E. coli with a limit of detection of 1.1CFU/mL and a linear detection range from 102 to 108CFU/mL, showing superior sensitivity compared to other reported methods. The technique also showed recovery value ranging from 83 to 125% for lettuce sample. The platform combines the advantages of microfluidics and SERS, enabling rapid, sensitive, and label-free detection of pathogens in food. This method addresses limitations of conventional culture-based techniques and other molecular diagnostics, offering a promising tool for microbial food safety analysis.

Adapting single-walled carbon nanotube-based thin-film transistors to flexible substrates with electrolyte-gated configurations using a versatile tri-layer polymer dielectric.

Flexibility has been a key selling point in the development of carbon-based electronics and sensors with the promise of further development into wearable devices. Semiconducting single-walled carbon nanotubes (SWNTs) lend themselves well to applications requiring flexibility while achieving high-performance. Our previous work has demonstrated a tri-layer polymer dielectric composed of poly(lactic acid) (PLA), poly(vinyl alcohol) with cellulose nanocrystals (PVAc), and toluene diisocyanate-terminated poly(caprolactone) (TPCL), yielding an environmentally benign and solution-processable n-type thin-film transistor (TFT). Despite the potential for fabrication on flexible substrates, these devices were only characterized on rigid substrates. We present herein the fabrication of these TFTs on Kapton® substrates and a progression of the devices' n- and p-type operation over 7 days, demonstrating continuous loss of the n-type performance and relative stability of the p-type performance after 3 days in ambient air. The tri-layer dielectric is then applied in an electrolyte-gated SWNT field-effect transistor (EG-SWNT-FET) architecture, shielding the SWNTs from the electrolyte and allowing for width-normalised g m values of 0.0563 ± 0.0263 μS μm-1 and I ON/OFF ratios of 103-104 using de-ionized (DI) water as the electrolyte. Finally, as a proof of concept, the device was used to detect α-synuclein, a neuronal protein whose aggregation is associated with Parkinson's disease, in DI water through the immobilization of target specific aptamer molecules on the polymer layer covering the gate electrode.

Evaluating the efficacy of specific aptamers for post-translational modified hemoglobin A by developing a lateral flow assay based on fluorescent carbon dot signals and smartphone-based read-out with quantification application software

Evaluating the efficacy of specific aptamers for post-translational modified hemoglobin A by developing a lateral flow assay based on fluorescent carbon dot signals and smartphone-based read-out with quantification application software

Competitive Electrochemical Apta-Assay Based on cDNA-Ferrocene and MXenes for Staphylococcus aureus Surface Protein A Detection.

Staphylococcus aureus (S. aureus) represents one of the most frequent worldwide causes of morbidity and mortality due to an infectious agent. It is a part of the infamous ESKAPE group, which is highly connected with increased rates of healthcare-associated infections and antimicrobial resistance. S. aureus can cause a large variety of diseases. Protein A (PrA) is a cell-wall-anchored protein of S. aureus with multiple key roles in colonization and pathogenesis and can be considered as a marker of S. aureus. The development of aptasensors, having an aptamer as a specific biorecognition element, increases selectivity, especially when working with complex matrices. The association with state-of-the-art materials, such as MXenes, can further improve the analytical performance. A competitive aptasensor configuration based on a ferrocene (Fc)-labeled cDNA hybridized (cDNA-Fc S13) on a specific aptamer (APT) for PrA in the presence of MXene nanosheets was designed for the indirect detection of S. aureus. The aptasensor displayed a linear range of 10-125 nM, an LOD of 3.33 nM, and a response time under 40 min. This configuration has been tested in real samples from volunteers diagnosed with S. aureus infections with satisfactory results, enabling the perspective to develop decentralized devices for the rapid detection of bacterial strains.

Tailoring Infrared Light-Molecule Coupling for Highly Sensitive Cortisol Detection Employing Aptamer-Conjugated Gold Nanonails.

Chemically synthesized gold nanoantennas possess easy processability, low cost, and suitability for large-area fabrication, making them advantageous for surface-enhanced infrared (SEIRA) biosensing. Nevertheless, current gold nanoantennas face challenges with limited enhancement of biomolecular signals that hinder their practical applications. Here, we demonstrate that the coupling rate between antennas and molecules critically impacts the enhancement of molecular signals based on temporal coupled mode theory. To improve this coupling rate, we synthesized gold nanonails with sharp tips, significantly amplifying the localized electric fields of antenna resonance modes. Modulating the nanonail aspect ratio allows us to tailor antenna resonance frequencies to match molecular vibrational frequencies. Additionally, we introduced specific aptamers on antenna surfaces through solution exchange methods to control the antenna-molecule distances. These combined strategies enabled noninvasive, label-free detection with high sensitivity for the biomarker cortisol. Experiments revealed 3 orders of magnitude enhancement in cortisol detection levels upon increasing coupling efficiency, achieving a detection limit of 0.1 ng/mL, notably lower than the normal cortisol concentration in human saliva (0.398 ng/mL). In addition to demonstrating a novel strategy for cortisol detection, this study provides a viable approach to biomarker detection for future applications in disease diagnosis and human health monitoring.

A simple lable-free aptasensor based on liquid exfoliated graphene modified electrode for chloramphenicol detection in milk

Antibiotic residues in animal derived foods considered as one of the most serious food contamination poses significant risks to human health. Conventional methods for antibiotic residue detection rely on expensive and bulky instruments, complicated sample pretreatment and time-consuming, thus limiting their applications. Consequently, developing miniaturized and low-cost analytical technology for ultrasensitive monitoring and accurate evaluation of antibiotic residues in foods is of great significance. Herein, a simple and label-free electrochemical sensing platform for the highly sensitive detection of chloramphenicol (CAP) in milk was demonstrated by employing aptamer specific for CAP as a biological recognition element and liquid exfoliated graphene (LEG) modified indium tin oxide electrode (LEG/ITO) as substrate. LEG is believed to be the most economic few-layer graphene with remarkable electrical properties and larger specific surface area not only promotes electron transfer but also provides a large number of binding sites for aptamers to be assembly immobilized via the π–π stacking of DNA bases. Under optimal experimental factors, the aptasensor displayed a wider linear range for detecting CAP (1 fM ∼ 100 nM), accompanied by a limit of detection (LOD) (1 fM), as well as satisfactory performance with specificity, reproducibility, and stability. In addition, the designed strategy allowed the direct analysis of actual milk samples and the recovery rates were from 97.92 % to 103.34 %, rendering a ideal sensing strategy for quantitative determination of various analytes in food by adapting the specific aptamer.

DNA tweezers aptasensor with split aptamer for the dual detection of Enrofloxacin and Kanamycin

Currently, the technology for detecting antibiotics is developing rapidly, with good sensitivity and selectivity. However, some detection methods still have issues with cumbersome pretreatment and high experimental costs. Herein, A “turn-off” bifunctional DNA tweezer was constructed through the design of DNA sequence and self-assembly technology for the dual detection of Enrofloxacin (ENR) and Kanamycin (KANA) in a food matrix. The operation is simple, which increases the detection speed and reduces the detection cost. The two ends of the bifunctional DNA tweezers are connected with specific split aptamer sequences, and the split aptamer forms a specific ternary complex with the target. The tweezers “clip” ENR and KANA at their respective ends, causing a structural change. Under the optimal experimental conditions, the linear range of detection ENR is 0.2 ng/mL-1000 ng/mL with an LOD of 0.18 ng/mL, the linear range of detection KANA is 0.5 ng/mL-1000 ng/mL with an LOD of 0.36 ng/mL. The bifunctional DNA tweezers involved provide a new idea for the construction of DNA nanomachines, which is also suitable for the recognition and detection of other small molecules.

Wearable Aptasensors.

This Perspective explores the revolutionary advances in wearable aptasensor (WA) technology, which combines wearable devices and aptamer-based detection systems for personalized, real-time health monitoring. The devices leverage the specificity and sensitivity of aptamers to target specific molecules, offering broad applications from continuous glucose tracking to early diagnosis of diseases. The integration of data analytics and artificial intelligence (AI) allows early risk prediction and guides preventive health measures. While challenges in miniaturization, power efficiency, and data security persist, these devices hold significant potential to democratize healthcare and reshape patient-doctor interactions.

Inhibitory DNA Aptamer Tools against SOCS3-gp130 Interactions.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a crucial role in transmembrane signal transduction, enabling cells to communicate with the extracellular environment, which requires precise regulation to prevent abnormal signaling outcomes. Within this complex pathway, suppressor of cytokine signaling 3 (SOCS3) acts as a pivotal negative regulator via binding to cytokine receptors. However, our understanding of the regulatory mechanisms governing SOCS3 interactions has been limited by the lack of suitable chemical probes. Aptamers, a notable category of chemical tools, have emerged as a viable avenue for the development of chemical inhibitors. In this study, we successfully identified specific inhibitory aptamers that bind to SOCS3 and effectively disrupt the interaction between SOCS3 and glycoprotein 130, a common cytokine receptor for interleukin-6 and leukemia inhibitory factor, which regulates the survival of normal human endometrial stromal cells. These aptamers not only provide a valuable chemical tool to advance our understanding of the regulatory dynamics of the JAK/STAT signaling pathway by SOCS3 but also hold great promise for the future development of therapeutic interventions.

A facile optical sensing strategy for glyphosate detection based on structure-switching signaling aptamers.

Afacile and highly specific optical sensing strategy is established for glyphosate (GLYP) detection using structure-switching signaling aptamers (F-SSSAs) with fluorescence signal reporting functionality. The strategy involves two domains: the FITC-labeled signal transduction domain for fluorescence signal reporting, while the functional domain (specific structure-switching aptamers) controls the target recognition. Graphene oxide (GO) works as a robust F-SSSAs quencher in the absence of GLYP. However, the F-SSSAs structure is switched in the presence of GLYP, prominently affecting the interaction with GO. The fluorescence of the structure-switching signaling aptamer-based sensing system is subsequently restored. The present strategy exhibits two dynamic linear relationships for GLYP detection in the ranges 0.2 to 80 ng·mL-1 and 100 to 800 ng·mL-1, with a low detection limit (LOD) of 0.07 ng·mL-1. Significantly, the proposed sensing system has been successfully utilized to detect GLYP in water, soil, and rice, demonstrating its potential applications in GLYP monitoring.

A polydopamine coated magnetic spiral switchable composite material for photothermal sterilization and dual-color fluorescence detection of food-borne pathogens

A multifunctional magnetic composite material (MSp@PDA) featuring with helical structure, superparamagnetism, photothermal properties, and fluorescence quenching capability was synthesized through sequential magnetization and in-situ polydopamine coating on the Spirulina surface, then applied for multi-scenario application of pathogens capture, sterilization, and sensitive detection. The pathogens capture and sterilization was investigated by loading bacterial broad-spectrum recognition molecule (MPBA) on MSp@PDA, the capture efficiencies of 91.6 % and 94.2 % were obtained for Escherichia coli and Staphylococcus aureus in 15 minutes, respectively, with complete photothermal sterilization achieved within 4 minutes. The fluorescence real-time detection of E. coli and S. aureus was realized by connecting dual-color fluorescent carbon dots to MSp@PDA through specific aptamers (dCD-apt-MSp@PDA), the corresponding detection limits were 2 and 3 cfu·mL−1, respectively, with linear ranges of 101∼107 cfu·mL−1. The spiked recovery rates of target bacteria in actual samples were 97.4∼101.1 % and 98.2∼101.4 % for E. coli and S. aureus, respectively. The constructed capture, sterilization, and fluorescence detection methods demonstrated promising application value for on-site monitoring of food-borne pathogens in complex matrices.

Varieties of interactions of anti-CD133 aptamers with cell cultures from patient glioblastoma

Development of aptatheranostics for glioblastoma (GB) requires investigating aptamer interactions with cells. The paper has described flow cytometry (FC) assessment of direct interactions of fluorescent anti-CD133 aptamers with cells, focusing on cell cultures derived from patient GB (CCPGB). Conventional cell lines with different levels of CD133 mRNA, Caco-2 and HCT116, were used to compare interactions with known 2'FY-RNA aptamer A15 and DNA aptamers of Ap and Cs series, labeled with FAM and Cy5. In addition, interactions of certain non-aptameric oligonucleotides were studied. In the case of antibody interactions with cells, FC signals, mean fluorescence intensities (MFIs), correlated with sizable amounts of CD133 mRNA in Caco-2 cells, and CCPGBs 107 and G01. Unexpectedly, MFI per se could not be the solid indicator of specific interactions of aptamer - CD133/cell. Instead, two types of interactions, target CD133-driven and off-target membrane-associated ones, contribute to MFI. The latter was notably observed for CCPGB Sus/fP2 with tiny CD133 mRNA amount. To prove specificity of aptamer - CD133/cell interactions, titration experiments have been performed, revealing half-saturation concentrations of 120±27 for 2'FY-RNA A15 and 180±12 for DNA Cs5 with Caco-2 cells. This knowledge is an essential step to develop aptatheranostics for GB.

Research progress on bone repair biomaterials with the function of recruiting endogenous mesenchymal stem cells

To review the research progress on bone repair biomaterials with the function of recruiting endogenous mesenchymal stem cells (MSCs). An extensive review of the relevant literature on bone repair biomaterials, particularly those designed to recruit endogenous MSCs, was conducted, encompassing both domestic and international studies from recent years. The construction methods and optimization strategies for these biomaterials were summarized. Additionally, future research directions and focal points concerning this material were proposed. With the advancement of tissue engineering technology, bone repair biomaterials have increasingly emerged as an ideal solution for addressing bone defects. MSCs serve as the most critical "seed cells" in bone tissue engineering. Historically, both MSCs and their derived exosomes have been utilized in bone repair biomaterials; however, challenges such as limited sources of MSCs and exosomes, low survival rates, and various other issues have persisted. To address these challenges, researchers are combining growth factors, bioactive peptides, specific aptamers, and other substances with biomaterials to develop constructs that facilitate stem cell recruitment. By optimizing mechanical properties, promoting vascular regeneration, and regulating the microenvironment, it is possible to create effective bone repair biomaterials that enhance stem cell recruitment. In comparison to cytokines, phages, and metal ions, bioactive peptides and aptamers obtained through screening exhibit more specific and targeted recruitment functions. Future development directions for bone repair biomaterials will involve the modification of peptides and aptamers with targeted recruitment capabilities in biological materials, as well as the optimization of the mechanical properties of these materials to enhance vascular regeneration and adjust the microenvironment.

Rapid detection of Brucella cells using a gold nanoparticle-based aptasensor via a simple colorimetric method

BackgroundBrucellosis is a major worldwide zoonotic disease that is caused by Brucella spp. and threatens the health of communities. Novel methods for rapid detection of Brucella bacteria are beneficial and necessary in preventing infection and subsequent economic losses. Constructing biosensors with nanoparticles is a promising approach for identification of pathogenic bacteria in a short time. This study aimed to introduce a new detection method of Brucella cells using a biosensor, based on gold nanoparticles and a specific aptamer, via a colorimetric reaction. In this work, gold nanoparticles (GNPs) were synthesized and attached to the aptamer through electrostatic bonding. The binding of aptamer to gold nanoparticles was confirmed by Uv/vis spectrophotometry, FT-IR, transmission electron microscope (TEM) and zeta sizer (DLS).ResultsIn the presence of the bacterial cells, aptamers were bound to their targets, and the surfaces of the nanoparticles were depleted from aptamers resulting in intensified peroxidation activity of GNPs, and with the addition of 3, 3′, 5, 5′-tetramethylbenzidine (TMB), the color of the solution was changed from red to purple, which indicated the presence of Brucella. The sensitivity of the aptasensor was investigated using different concentrations of Brucella cells and its specificity was confirmed against several species of bacteria. The results showed that the designed aptasensor was more sensitive compared to PCR assay method with the ability to detect 1.5 × 101 CFU/mL of the bacterial cells.ConclusionThese findings indicate that the designed aptasensor can be used as a simple and rapid diagnostic tool to detect Brucella cells without need to experts and expensive laboratory equipment.

Visible light-activated signal amplification PEC aptasensor for ultrasensitive zearalenone detection based on double Z-type BiOI/g-C3N4@Au/Bi2S3 heterojunctions

Zearalenone (ZEN), also known as F-2 toxin, can enter the body through the food chain and accumulate, leading to DNA break and an abnormal number of chromosomes. Therefore, it is crucial to develop a facile and sensitive method for detecting ZEN. Herein, a visible light-activated signal amplification photoelectrochemical (PEC) aptasensor for ultrasensitive detection of ZEN has been successfully constructed, exploiting the synergistic effect of the Z-type BiOI/g-C3N4/Au heterojunctions, Bi2S3 as a signal enhancer, and the remarkable affinity and specificity of aptamers. The PEC properties and electron transfer mechanism of the double Z-type heterojunctions were thoroughly investigated. When the aptamers captured the ZEN molecules, Bi2S3 and cDNA departed from the electrode, resulting in a reduction in the photocurrent signal. Therefore, the concentration of ZEN can be quantitatively analyzed by measuring the change in the photocurrent response. The constructed PEC aptasensor exhibited exceptional sensitivity and selectivity for ZEN in the concentration range of 0.1 fg⋅mL−1 ––1.0 ng⋅mL−1, with a detection limit of 0.087 fg⋅mL−1 (S/N = 3). Finally, the PEC aptasensor was successfully applied to detect ZEN in actual coix seed samples, providing some guidance for the development of PEC aptasensors for practical medicine monitoring.

Detection of C-reactive protein using a label-free NIR fluorescent aptasensor with a large Stokes shift based on an AIEE anthracene derivative

BackgroundC-reactive protein (CRP), one of the classic biomarkers of inflammation, is closely related to infectious inflammation, cardiovascular disease, cancer, and other diseases. Therefore, timely and accurate detection of CRP in human blood is crucial for the discovery, diagnosis, and treatment of the aforementioned diseases. Herein, a novel label-free NIR fluorescence aptasensor with a large Stokes shift based on an AIEE anthracene derivative B and a molybdenum disulfide (MoS2) platform was developed and used for the high sensitivity and specificity detection of CRP. ResultsCompound B could emit near-infrared (NIR) fluorescence with a large Stokes shift (190 nm). Notably, this compound could bind with the aptamer of CRP (CRP-Apt) through electrostatic attraction to form a B/CRP-Apt complex, generating an aggregation-induced emission enhancement effect and enhancing the fluorescent intensity of B. B/CRP-Apt could be adsorbed on the surface of MoS2 with the addition of MoS2 to its solution, and the fluorescence of Compound B was quenched. CRP was then added to the above solution. CRP-Apt had a substantially higher affinity for CRP than MoS2. Therefore, B/CRP-Apt detached from the surface of MoS2 and bound to CRP, thereby restoring the fluorescence of B. Experimental results showed a good linear relationship between the fluorescent recovery intensity of B and the concentration of CRP in the concentration range of 0.3–70 ng mL−1, with a limit of detection as low as 0.1 ng mL−1. Significance and noveltyThe aptasensor integrates the advantages of high sensitivity of NIR fluorescence, high specificity of aptamers, good water-solubility and AIEE effect of Compound B. And it could be applied to the determination of CRP in human serum samples, while most of the reported methods can only determine CRP in spiked human serum samples.

Integrating All-rounder TiO2 Accelerated Electrochemiluminescence with Dual-Quenching PDA@COF Probes for Sensitive Quantification and Protein Profiling of Tumorous Exosomes.

Exosomes have been perceived as promising biomarkers for noninvasive cancer diagnosis and treatment monitoring. However, the sensitive and accurate quantification and phenotyping of exosomes remains challenging. Herein, a versatile electrochemiluminescence (ECL) aptasensor was proposed for the sensitive analysis of tumorous exosomes. Specifically, a ternary nanohybrid (Ru-HAuTiO2), by covalently linking ECL luminophore Ru(dcbpy)32+ with gold nanoparticles (AuNPs)-decorated hollow urchin-like TiO2 (HTiO2), was ingeniously designed as a highly luminescent and self-enhanced ECL nanoemitter. Notably, the porous HTiO2 played an "all-rounder" role, including the carrier for ECL luminophores and AuNPs, coreaction accelerator, and specific exosome capturing scaffold through Ti-phosphate coordination interaction. On the other hand, a polydopamine modified covalent organic framework (PDA@COF) was employed as a quencher to remarkably attenuate the ECL of Ru-HAuTiO2 through a dual-quenching mechanism, and further labeled with a specific aptamer (Apt) of exosomal surface protein. Based on forming a Ru-HAuTiO2/exosome/Apt-PDA@COF sandwich structure on the electrode, a "signal on-off" ECL platform for tumorous exosomes was constructed, realizing sensitive detection within the range of 3.1 × 103 particles/mL to 1 × 108 particles/mL and a low limit of detection of 1.41 × 103 particles/mL, achieving phenotypic profiling of surface proteins on different tumorous exosomes. This work provides a promising alternative method for the detection and analysis of exosomes.

Selection of novel ssDNA aptamer for ultra-sensitive colorimetric detection of acetamiprid using ultra-small dot assay

An innovative ssDNA aptamer-mediated colorimetric biosensor utilizing an ultra-small dot assay has been developed for the ultra-sensitive detection of acetamiprid, a hazardous insecticide widely used in agriculture. This “Dot assay” leverages ultra-low volumes of less than 20 µL, allowing for rapid visual detection within seconds and addressing the urgent need for accessible and efficient detection methods. Through a non-immobilized graphene oxide-based systematic evolution of ligands by exponential enrichment (SELEX) approach, a specific 79-mer aptamer (Apt-SS5) was identified after 15 enrichment cycles. The Apt-SS5 aptamer demonstrated a remarkable binding affinity for acetamiprid, with a melting temperature (Tm) of 53 °C and a Gibbs free energy (ΔG) of −7.24 kcal/mol at 37 °C. Notably, the binding pocket is formed by nucleotides C-16 to T-32, with critical interactions occurring at C-10, C-16, G-25, A-26, G-27, G-28, and A-29. The optimized colorimetric assay achieved an impressive limit of detection (LOD) of 0.039 ppb and resulted in a significant reduction in assay costs. The target specificity and cross-reactivity of Apt-SS5 were tested against other pesticides, including imidacloprid, thiachloprid, thiamethoxam, chlorpyrifos, pyriproxyfen, and chlorantraniliprole, with no significant color change observed for these non-targets. In contrast, a clear visual color change was noted in the presence of acetamiprid, confirming the aptamer’s high specificity. Additionally, the aptasensor proved effective for testing river and water samples, yielding recovery rates of 84.40 % to 101.66 %. This innovative approach enhances sensitivity and simplifies the detection process, paving the way for rapid screening of pesticide residues in various samples.