Aptamer Sequence Research Articles

Aptamer-based protein molecule detection via cyclic reverse transcription coupling with self-priming hairpin-triggered CRISPR-Cas12a system

Protein biomarkers (e.g. thrombin) are of great significance for the biological process of the organism, and its aberrant expression is closely associated with the development of diseases. With thrombin, a serine protease that plays a crucial role in maintaining homeostasis and promoting blood clotting, as detection target, this study introduces a novel approach for sensitive and accurate measurement of protein biomarker expression by utilization of cyclic reverse transcription (CRT) in combination with the self-priming hairpin-triggered CRISPR-Cas12a system. In this method, an elegantly designed sensing probe is utilized to specifically bind with the thrombin protein and convert the protein signals to nucleic acids signals, following by the CRT and CRISPR-Cas12a system-based signal amplification strategy. Taking the merit of the two-stage amplification, this assay has the capability to detect thrombin at the fM level. In addition, due to the aptamer sequence’s strong selectivity to thrombin protein and the dual-check process in the signal amplification process (first in the CRT and second in the CRISPR system), the proposed test demonstrates exceptional specificity in detecting thrombin. By re-designing the sensing probe, the established method could be extended to various protein biomarker detection. Ultimately, this assay has successfully enabled the accurate evaluation of biomarker levels in constructed clinical samples, showing significant potential for application in the realm of clinical molecular diagnosis.

High-density aptamer synthesis based on primer exchange reaction for label-free electrochemical biosensing

We report an electrochemical aptamer-based (E-AB) sensor for label-free quantitative detection of thrombin. In-situ synthesis of high-density aptamer replicas on the electrode is accomplished based on primer exchange reaction (PER), by which a DNA primer is continuously extended with the presence of a catalytic hairpin template and DNA polymerase. The PER enables the synthesis of long single-stranded DNA with replicated aptamer sequence for thrombin binding. The PER products on the surface of a gold electrode increase the density of aptamers on a limited electrode area, which can provide more effective binding sites for thrombin and therefore improve the performance of the sensor based on electrochemical impedance spectroscopy. Compared with the conventional E-AB sensor based on a 15-mer aptamer immobilized on the electrode, our method has a lower detection limit of 0.91 pM, and a wider linear range of 1.0 pM to 50 nM. Therefore, we believe our method is promising for label-free electrochemical detection of biomarkers.

Target and acidic tumor microenvironment-induced in situ HCR for bispecific tumor cell imaging and therapy

The tumor microenvironment is characterized by an acidic extracellular milieu generated through the anaerobic glycolysis of tumor cells. Leveraging this characteristic, we suggest employing an in situ hybridization chain reaction (HCR) activated by both target recognition and the acidic microenvironment for bispecific tumor cell imaging and therapy. By integrating the recognition capability of aptamers, the pH responsiveness of i-motifs, and the signal amplification of HCR, we have developed a pH-induced HCR (pH-HCR) system. This system consists of three DNA probes: pH-Apt, HP1, and HP2. pH-Apt is composed of the Apt-T-B and Imotif sequences (sequences that can form an i-motif structure). Apt-T-B includes two segments: an aptamer sequence for MUC1 recognition and a trigger sequence blocked by the Imotif sequence for HCR. Under acidic conditions, upon binding to MUC1, the Imotif sequence forms an i-motif structure, detaching from pH-Apt and exposing the trigger sequence. This initiation of HCR, in conjunction with HP1 and HP2, amplifies fluorescent signals, achieving the bispecific imaging of tumor cells. Simultaneously, doxorubicin (Dox) can be loaded onto probes and released under acidic conditions, resulting in a 47% cytotoxic efficiency against tumor cells. pH-HCR enables successful bispecific imaging and cell killing induced by MUC1 in the acidic microenvironment of SW620 cells.

Low-background fluorescent biosensor based on graphene oxide and aptamer biorecognition for sensitive detection of kanamycin

The detection of kanamycin has attracted extensive attention in the field of food safety. A label-free, low-background fluorescent biosensor was successfully constructed based on graphene oxide and aptamer biorecognition in this paper for kanamycin detection in milk samples. In this biosensor, the integrated probe with the aptamer sequence of kanamycin and the G-rich sequence was designed. The aptamer sequence of kanamycin was used as the recognition part, the G-quadruplex/N-methyl mesoporphyrin IX (NMM) complex as the fluorescent probe, and graphene oxide as the fluorescent quencher. Initially, the integrated probe could be adsorbed on the graphene oxide surface through π-π stacking interactions, while the fluorescence of NMM could be efficiently quenched because of the resonance energy transfer between NMM and graphene oxide, which was beneficial to the low background signal of the system. During the detection process, the aptamer sequence preferred to bind to kanamycin, forming aptamer-kanamycin complex. At the same time, the G-rich sequence folded into G-quadruplex structure that specifically bound to NMM under the action of K+, forming G-quadruplex/NMM complex. Under the synergistic effects of the two complexes, the integrated probe with aptamer-kanamycin complex and G-quadruplex/NMM complex was released from the graphene oxide surface due to the steric hindrance effect, restoring the strong fluorescence signal. By combining graphene oxide and G-quadruplex/NMM complex, the fluorescent biosensor could effectively achieve label-free and low-background detection of kanamycin. The linear calibration curve was in the range of 1–400 nM with a detection limit of 0.60 nM for kanamycin. It was successfully applied to analyze raw milk samples (spiked kanamycin) with recoveries of 98.33–101.25 % and also exhibited high selectivity for kanamycin. The experimental data showed that the biosensor was a potentially powerful tool for monitoring food safety.

Polyvalent Aptamer-Functionalized NIR-II Quantum Dots for Targeted Theranostics in High PD-L1-Expressing Tumors.

Ag2S quantum dots (QDs) show superior optical properties in the NIR-II region and display significant clinical potential with favorable biocompatibility. However, inherent defects of low targeting and poor solubility necessitate practical modification methods to achieve the theranostics of Ag2S QDs. Herein, we used rolling circle amplification (RCA) techniques to obtain long single-stranded DNA containing the PD-L1 aptamer and C-rich DNA palindromic sequence. The C-rich DNA palindromic sequences can specifically chelate Ag2+ and thus serve as a template to result in biomimetic mineralization and formation of pApt-Ag2S QDs. These QDs enable specific targeting and illuminate hot tumors with high PD-L1 expression effectively, serving as excellent molecular targeted probes. In addition, due to the high NIR-II absorption of Ag2S QDs, pApt-Ag2S QDs exhibit remarkable photothermal properties. And besides, polyvalent PD-L1 aptamers can recognize PD-L1 protein and effectively block the inhibitory signal of PD-L1 on T cells, enabling efficient theranostics through the synergistic effect of photothermal therapy and immune checkpoint blocking therapy. Summary, we enhance the biological stability and antibleaching ability of Ag2S QDs using long single-stranded DNA as a template, thereby establishing a theranostic platform that specifically targets PD-L1 high-expressing inflamed tumors and demonstrates excellent performance both in vitro and in vivo.

Adsorption of DNA and Aptamers to Sodium Urate Crystals and Inhibition of Crystal Growth.

An elevated level of blood uric acid (UA) can cause the formation of kidney stones, gout, and other diseases. We recently isolated a few DNA aptamers that can selectively bind to UA. In this work, we investigated the adsorption of a UA aptamer and random sequence DNA onto sodium urate crystals. Both DNA strands adsorbed similarly to urate crystals. In addition, both the UA aptamer and random DNA can inhibit the growth of urate crystals, suggesting a nonspecific adsorption mechanism rather than specific aptamer binding. In the presence of 500 nM DNA, the growth of needle-like sodium urate crystals was inhibited, and the crystals appeared granular after 6 h. To understand the mechanism of DNA adsorption, a few chemicals were added to desorb DNA. DNA bases contributed more to the adsorption than the phosphate backbone. Surfactants induced significant DNA desorption. Finally, DNA could also be adsorbed onto real UA kidney stones. This study provides essential insights into the interactions between DNA oligonucleotides and urate crystals, including the inhibition of growth and interface effects of DNA on sodium urate crystals.

Selection of ssDNA aptamers and construction of aptameric electrochemical biosensor for the detection of Giardia intestinalis trophozoite protein

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.

Chronoamperometric interrogation of an electrochemical aptamer-based sensor with tetrahedral DNA nanostructure pendulums for continuous biomarker measurements

Tetrahedral DNA nanostructure (TDN) is highly promising in developing electrochemical aptamer-based (E-AB) sensors for biomolecular detection, owing to its inherit programmability, spatial orientation and structural robustness. However, current interrogation strategies applied for TDN-based E-AB sensors, including enzyme-based amperometry, voltammetry, and electrochemical impedance spectroscopy, either require complicated probe design or suffer from limited applicability or selectivity. In this study, a TDN pendulum-empowered E-AB sensor interrogated by chronoamperometry for reagent-free and continuous monitoring of a blood clotting enzyme, thrombin, was developed. TDN pendulums with extended aptamer sequences at three vertices were immobilized on a gold electrode via a thiolated double-stranded DNA (dsDNA) at the fourth vertex, and their motion is modulated by the bonding of target thrombin to aptamers. We observed a significantly amplified signalling output on our sensor based on the TDN pendulum compared to E-AB sensors modified with linear pendulums. Moreover, our sensor achieved highly selective and rapidly responsive measurement of thrombin in both PBS and artificial urine, with a wide dynamic range from 1 pM to 10 nM. This study shows chronoamperometry-enabled continuous biomarker monitoring on a sub-second timescale with a drift-free baseline, demonstrating a novel approach to accurately detect molecular dynamics in real time.

One-pot, one-step, label-free miRNA detection method based on the structural transition of dumbbell probe

In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0–10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.

A nitrogen-doped hollow carbon nanospheres-based aptasensor for non-invasive salivary detection of progesterone

Developing an easy-to-use and non-invasive sensor for monitoring progesterone (P4) as a multi-functional hormone is highly demanded for point-of-care testing. In this study, an ultrasensitive electrochemical aptasensor is fabricated for monitoring P4 in human biofluids. The sensing interface was designed based on the porous nitrogen-doped hollow carbon spheres (N-HCSs). The N-HCSs covalently immobilized high-dense aptamer (Apt) sequences as the bioreceptor of P4. The electron transfer of the redox probe was hindered by incubating P4 on the aptasensor surface and forming the P4-Apt complexes. Meanwhile, the signaling was decreased under two wide linear dynamic ranges (LDRs) from 10 fM to 5.6 μM with a limit of detection (LOD) value of 3.33 fM. The aptasensor presented satisfactory selectivity in the presence of different off-target species with successful feasibility for P4 detection in some human urine and saliva samples. The aptasensor with high sensitivity, as an advantage for on-site and sensitive measurement of P4, can be considered a non-invasive tool for routine analysis of real-world clinical samples method.

Enrofloxacin Rapid Detection in Aquatic Foods: Based on DNA Aptamer Sensor.

Enrofloxacin (ENR) is widely used as a synthetic fluoroquinolone antibiotic for disease control in aquatic animals. ENR aptamers were screened in this study using the magnetic bead-SELEX method, and a graphene oxide fluorescent sensor was developed to detect the ENR residues in aquatic products. Firstly, ENR was conjugated to amino magnetic beads by amidation reaction, and then the aptamer sequences showing high affinity to ENR were screened step by step by using the SELEX screening method. Finally, after 10 rounds of SELEX screening, six candidate aptamers with high affinity were obtained. Among these, ENR-Apt 6 was selected based on its secondary structure features, high affinity (Kd = 35.08 nM), and high specificity to ENR. Furthermore, a fluorescent sensor was prepared using graphene oxide and ENR-Apt 6. The results showed that the linear range of the sensor could reach 600 nM (R2 = 0.986), while its optimal linear range was 1-400 nM (R2 = 0.991), with the lowest detection limit of 14.72 nM. The prepared sensor was successfully used for the detection of ENR in real samples, with a recovery range of 83.676-114.992% and a relative standard deviation < 10% for most of the samples.

DNA Origami-Engineered Plasmonic Nanoprobes for Targeted Cancer Imaging.

Plasmonic nanomaterials bearing targeting ligands are of great interest for surface-enhanced Raman scattering (SERS)-based bioimaging applications. However, the practical utility of SERS-based imaging strategies has been hindered by the lack of a straightforward method to synthesize highly sensitive SERS-active nanostructures with high yield and efficiency. In this work, leveraging DNA origami principles, we report the first-in-class design of a SERS-based plasmonically coupled nanoprobe for targeted cancer imaging (SPECTRA). The nanoprobe harnesses a cancer cell targeting DNA aptamer sequence and vibrational tag with stretching frequency in the cell-silent Raman window. Through the integration of aptamer sequence specific for DU145 cells, we show the unique capabilities of SPECTRA for targeted imaging of DU145 cells. Our results demonstrate that the scalability, cost-effectiveness, and reproducibility of this method of fabrication of SERS nanoprobes can serve as a versatile platform for creating nanoprobes with broad applications in the fields of cancer biology and biomedical imaging.

Aptamer-mediated double strand displacement amplification with microchip electrophoresis for ultrasensitive detection of Salmonella typhimurium

Rapid and quantitative detection of foodborne bacteria is of great significance to public health. In this work, an aptamer-mediated double strand displacement amplification (SDA) strategy was first explored to couple with microchip electrophoresis (MCE) for rapid and ultrasensitive detection of Salmonella typhimurium (S. Typhimurium). In double-SDA, a bacteria-identified probe consisting of the aptamer (Apt) and trigger sequence (Tr) was ingeniously designed. The aptamer showed high affinity to the S. Typhimurium, releasing the Tr sequence from the probe. The released Tr hybridized with template C1 chain, initiating the first SDA to produce numerous output strands (OS). The second SDA process was induced with the hybridization of the liberated OS and template C2 sequence, generating a large number of reporter strands (RS), which were separated and quantified through MCE. Cascade signal amplification and rapid separation of nucleic acids could be realized by the proposed double-SDA method with MCE, achieving the limit of detection for S. typhimurium down to 6 CFU/mL under the optimal conditions. Based on the elaborate design of the probes, the double-SDA assisted MCE strategy achieved better amplification performance, showing high separation efficiency and simple operation, which has satisfactory expectation for bacterial disease diagnosis.

A highly sensitive ratiometric electrochemical aptasensor for prostate specific antigen detection based on toehold-mediated strand displacement reaction

In this work, a novel ratiometric electrochemical aptasensor was designed for prostate specific antigen (PSA) detection based on toehold-mediated strand displacement reaction (TMSDR). For PSA detection, PSA could bind with the aptamer sequence in hairpin probe 1 (HP1), and HP1/PSA complex was obtained. Then, in the presence of S3 DNA, HP1/PSA could trigger two TMSDR processes by hybridizing with Fc-S2/S1 double-stranded DNA, which would generate a large amount of Fc-S2 and recycle HP1/PSA. This enabled the hybridization of Fc-S2 with methylene blue labeled thiolated hairpin probe 2 (MB-HP2) immobilized on the electrode surface, causing MB molecules away from the electrode and Fc close to the electrode. Thus, with the increasing PSA concentrations, the current of MB (IMB) decreased and the current of Fc (IFc) increased. The ratio of IFc/IMB was shown to be linear with the logarithm of PSA concentration from 1 pg mL−1 to 100 ng mL−1, with a detection limit of 0.28 pg mL−1. In addition, the aptasensor had good specificity, reproducibility, repeatability, and stability for PSA detection, and was successfully applied to spiked diluted human serum samples.

Theranostic potential of a novel aptamer specifically targeting HER2 in breast cancer cells.

The overexpression of HER2 is correlated with poorer outcomes and therapeutic resistance in breast cancer patients. While HER2-targeted therapies have shown improvement, prognosis remains poor for HER2-positive breast cancer patients, and these treatments have limitations. Therefore, it is crucial to explore effective molecular strategies for early detection and treatment of HER2-positive breast cancers. In this study, we employed the cell-SELEX method to generate a selective aptamer capable of recognizing HER2 in its native conformation within breast cancer cells, for theranostic applications. Utilizing an adherent cell-SELEX approach, we developed and explored a DNA aptamer, named HMAP7, which can specifically target HER2 in the MDA-MB-453 and SK-BR-3 human breast cancer cell lines. After sequencing, the binding affinities of 10 candidate aptamers to HER2 receptors were evaluated by measuring fluorescence intensities within intact cells using near-infrared optical imaging. The dissociation constant of HMAP7 was determined to be in the nanomolar range in both cell lines. The cell-SELEX-derived aptamer sequence, HMAP7 (41-mer), exhibited the highest binding affinity and specificity for HER2. HMAP7 was rapidly internalized into breast cancer cells overexpressing HER2 but showed no uptake in the HER2 receptor-deficient breast cancer cell line MDA-MB-231. Moreover, HMAP7 demonstrated remarkable selectivity for HER2, rendering it suitable for use in complex biological systems. Our findings suggest that the novel DNA aptamer HMAP7 holds promise for both therapeutic and diagnostic applications, enabling selective delivery of therapeutic agents or imaging of HER2-positive breast tumors.

Analysis of human epidermal growth factor receptor 2 interaction on aptamer-probed interdigitated electrode for breast cancer diagnosis.

Breast cancer has been reported to be high in its incidence with women, and early identification of breast cancer helps to improve and provide an effective treatment. Tumor markers are active substances; in particular, human epidermal growth factor receptor 2 (HER2) is over-expressed at the level of 20%-30%. This research work developed a highly sensitive HER2 biosensor on the interdigitated electrode (IDE) by using aptamer as a detection probe. To enhance the analytical performances, aptamer was attached to the gold nanoparticle and immobilized on the IDE through a chemical linker [(3-aminopropyl)triethoxysilane]. On the aptamer conjugation, HER2 was quantified through current-volt measurements, and the limit of detection of HER2 was calculated as 1pg/mL on a linear range from 0.1 to 3000pg/mL at an R2 (regression coefficient) of 0.9657. Further, a selective performance with human serum increased the current responses by increasing HER2 concentrations. Specific experiments with control protein and complementary aptamer sequence failed to enhance the current responses. This HER2 biosensor reflects the occurrence of breast cancer at its lower abundance and helps to identify the associated complications.

Generation of epitope-specific hCG aptamers through a novel targeted selection approach.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which is usually specific to the C-terminal peptide of the β-subunit. However, cleavage of this region in many hCG degradation variants prevents rapid diagnostic tests from quantifying all hCG variants in serum and urine samples. An epitope contained within the core fragment, β1, represents an under-researched opportunity for developing immunoassays specific to most variants of hCG. In the study described here, we report on a SELEX procedure tailored towards the identification of two pools of aptamers, one specific to the β-subunit of hCG and another to the β1 epitope within it. The described SELEX procedure utilized antibody-blocked targets, which is an underutilized strategy to exert negative selection pressure and in turn direct aptamer enrichment to a specific epitope. We report on the first aptamers, designated as R4_64 and R6_5, each capable of recognising two distinct sites of the hCG molecule-the β-subunit and the (presumably) β1-epitope, respectively. This study therefore presents a new SELEX approach and the generation of novel aptamer sequences that display potential hCG-specific biorecognition.

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 μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu $$\\end{document}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.

A simple Label-Free aptasensor for acrylamide monitoring using a Four-Way arm junction structure of DNA strands

Herein, a sensitive and simple tag-free aptasensor was employed for acrylamide determination via the fluorescent dye SYBR Green I (SGI) with the role of signal indicator. The foundation of this platform was built upon the establishment of a four-way junction structure, which resulted from the hybridization between terminus regions of aptamer and complementary (CP) strands. The preferential interaction of the aptamer sequence with acrylamide led to the disintegration of the four-way junction structure into the single strands, and consequently, a significant decrease in the output fluorescence signal. Under the optimal conditions, the proposed aptasensor was shown to detect acrylamide, with low detection limit (LOD) of 0.28 μmol/L in the linear dynamic range of 1 to 60 μmol/L with high selectivity. The aptasensor possesses remarkable proficiency in detecting acrylamide levels within coffee samples, rendering it as a highly effective tool for ensuring the safety and quality control of food products.

A trivalent aptasensor by using DNA tetrahedron as scaffold for label-free determination of antibiotics

Owing to advantage in high sensitivity and fast response, aptamer based electrochemical biosensors have attracted much more attention. However, inappropriate interfacial engineering strategy leads to poor recognition performance, which ascribe to the following factors of immobilized oligonucleotide strand including steric hindrance, interchain entanglement, and unfavorable conformation. In this work, we proposed a DNA tetrahedron based diblock aptamer immobilized strategy for the construction of label-free electrochemical biosensor. The diblock aptamer sequence is composite of T-rich anchor domain and recognition domain, where T-rich domain enabling anchored on the edge of DNA tetrahedron via Hoogsteen hydrogen bond at neutral condition. The DNA tetrahedron scaffold offers an appropriate lateral space for target recognition of diblock aptamer. More importantly, this trivalent aptamer recognition interface can be regenerated by simply adjusting the pH environment to alkaline, resulting in the dissociation of diblock aptamer. Under the optimum condition, proposed electrochemical aptasensor manifested a satisfied sensitivity for aminoglycosides antibiotic, kanamycin with a limit of detection of 0.69 nM, which is 45-fold lower than traditional Au–S immobilization strategy. Moreover, the proposed aptasensor had also successfully been extended to ampicillin detection by changing the sequence of recognition domain in diblock aptamer. This work paves a new way for the rational design of aptamer-based electrochemical sensor.