Aptamer Sequence Research Articles

In silico Design of a Quadruplex RNA Aptamer Against Carcinoembryonic Antigen, and Evaluating its Potential as a Drug Delivery Vehicle

Background: Colorectal cancer is the third most common cancer worldwide, with carcinoembryonic antigen (CEA) being a key biomarker for tumor prognostic assessment. This study focused on the in silico development of an RNA aptamer capable of efficiently targeting the CEA biomarker, and evaluating its potential to act as a drug delivery vehicle of chemotherapeutic anticancer drugs. Methods: In this study, five G-quadruplex RNA aptamers were manually designed and docked with the CEA protein. The aptamer sequence with the best docking score was optimized through various mutations, and these mutant sequences underwent drug-aptamer docking using AutoDock vina, followed by further docking experiments with CEA, with and without the drug, using the Hdock server. The best aptamer-drug configuration and the entire (aptamers-drug)-CEA complex were then analyzed using molecular dynamics (MD) simulation. Results: Irinotecan exhibited the highest binding affinity with three mutant sequences compared to 5-FU and Raltitrexed, with the seq 3-5/Irinotecan complex showing the best configuration (score: -11.6). This complex also demonstrated a good binding affinity with the target CEA (HDOCK score: -393.07). Throughout 31.6 ns simulation, the RMSD plot of the (aptamer-drug)-CEA complex showed an average of 8.7 A°. The residues of interacting amino acids had lower RMSF than 1.4 Å, except for the ALA A0 (RMSF:1.74 Å). Rg plot showed minimal fluctuation, which were maintained between 19.2 Å and 20.2 Å. The mean SASA was in the range of 112.8–125.8 nm2. H-bonds were observed with an average of 7.3 hydrogen bonds. MD simulation results indicated that the (aptamers-drug)-CEA complex maintained a stable, low-flexibility, and relatively compact structure throughout the simulation. Conclusion: The study concludes that the designed quadruplex RNA aptamer can potentially target the CEA biomarker with good binding affinity and serves as an effective carrier for Irinotecan. However, in vitro and in vivo experiments are necessary for further validation.

Ultrastable NAC-Capped CdZnTe Quantum Dots Encapsulated within Dendritic Mesoporous Silica As an Exceptional Tag for Anti-Interference Fluorescence Aptasensor with Signal Amplification.

In this work, we explored the potential of thiol-capped CdZnTe quantum dots (QDs) as an exceptional signal tag for fluorescence aptasensing applications. Employing a one-pot hydrothermal approach, we modulated the terminal functional groups of CdZnTe QDs using l-cysteine (Lcys), 3-mercaptopropionic acid (MPA), and N-acetyl-l-cysteine (NAC) as ligands. Our comparative analysis revealed that NAC-capped CdZnTe QDs (NAC-CdZnTe QDs) exhibited superior anti-interference capabilities and storage stability across various temperatures, pH levels, and storage durations. Encouraged by these promising results, we further optimized the use of ultrastable NAC-CdZnTe QDs encapsulated in dendritic mesoporous silica nanoparticles (DMSN@QDs) as an exceptional tag for the development of an advanced anti-interference fluorescence aptasensor for aflatoxin B1 (AFB1) detection. The developed aptasensor using DMSN@QDs as signal tags achieved a remarkable signal amplification of approximately 10.2 fold compared to the NAC-CdZnTe QDs coated silica (SiO2@QDs) labeled fluorescence aptasensor. This aptasensor was able to detect AFB1 within a wide range of 1 pg mL-1 to 200 ng mL-1, achieving a limit of detection as low as 0.41 pg mL-1 (S/N = 3). Crucially, the specific binding affinity between the aptamer and the target enabled the aptasensor to be easily customized for various targets by simply replacing the aptamer sequence with the desired one. The exceptional potential of NAC-CdZnTe QDs, particularly when encapsulated in DMSNs, leads to the development of highly sensitive and selective anti-interference fluorescence aptasensors for various targets, thereby, paving the way for advancements in a diverse range of applications.

A novel red emission carbon quantum dots-based aptasensor for detection of potassium ions in vitro and in vivo and its application in plant imaging

The determination of potassium ion (K+) concentration in organisms is of particular importance. Herein, an efficient, sensitive, and eco-friendly fluorescence aptasensor for detecting potassium ions (K+) was designed based on the interaction of aptamer (K-Apt), complementary sequence of aptamer (C-Apt), red emission carbon quantum dots (R-CDs), and graphene oxide (GO). Red luminescent CDs modified with K+ aptamer(R-CDs@K-Apt) and C-Apt modified GO (C-Apt@GO) were used as the fluorescence donor and acceptor, respectively, in this aptasensor. K+ was detected sensitively using the fluorescence resonance energy transfer (FRET) method. The linear range of this aptasensor was 5.0 × 10−9–2.2 × 10−7 mol/L, and its detection limit was as low as 6.2 × 10−11 mol/L. Importantly, this aptasensor can be employed for quantitative K+ detection in serum because of the enhancement of its selectivity and biocompatibility by the modification of biomolecules including K-Apt and C-Apt. Furthermore, the aptasensor’s red emission at 625 nm enabled it to completely avoid the background fluorescence caused by chlorophyll in wheat seedlings, allowing it to display the K+ concentration of the wheat through plant imaging. These findings lay a foundation for the development of a novel detection technique for analyzing the distribution of K+ in the body fluids of animals and plants.

Application of MOF Nanoenzyme in Rapid Detection of Food Allergens System

Food allergies are a common immune response that can lead to serious health problems. Rapid and accurate detection of allergens in food is crucial for protecting people’s health and quality of life. In this study, β-lactoglobulin in milk serves as a model allergen, and nucleic acid aptamer-loaded MN patches are utilized as rapid capture elements. Moreover, metal organic frameworks loaded with complementary aptamer sequences are employed as signal amplification elements. The combination of these two elements establishes a rapid visualization detection system for allergens, employing specific microneedle array patches-MOF nanoenzymes. The results show that the combination of TMB+H2O2 +MOF-RC has the highest peroxidase like activity of 1.732. Compared to TMB+H2O2 +MOF, it has increased by 0.547. Meanwhile, the MN-MOF detection method has excellent specificity for the detection of β-Lg. This specific microneedle array patches and MOF nanoenzymes-based allergen rapid detection system demonstrates practical application benefits, providing a viable solution for swiftly detecting food allergens and safeguarding people’s health and quality of life.

Discovery and design of an aptamer that inhibits Shiga toxin type 2 activity by blocking Stx2 B subunit-Gb3 interaction

Shiga toxin (Stx) is the definitive virulence factor of Stx-producing Escherichia coli. This bacterial pathogen can contaminate food and threaten human health. Binding of the B subunit of Stx to the specific receptor globotriaosylceramide (Gb3) on the cell membrane is a key step for Stx to enter cells and exert its toxicity. In this work, we aimed to screen for aptamers targeting the Stx 2 B subunit, to interfere with the interaction of Stx2 B subunit and Gb3, thereby blocking Stx2 from entering cells. The results of molecular simulation docking, competitive ELISA, flow cytometry, and laser confocal microscopy confirmed that aptamers S4, S5, and S6 can mediate the interaction between Stx2 B subunit and Gb3. To further improve the inhibition effect, multiple aptamer sequences were tailored and were fused. The bivalent modification aptamer B2 inhibited Stx2 toxicity to Vero cells with inhibition rate of 53 %. Furthermore, the aptamer B2 reduced Stx2 damage to the mice, indicating that it has great potential to interfere with Stx2 binding to Gb3 receptors in vivo and in vitro. This work provides a theoretical and experimental basis for the application of aptamers in the inhibition of Stx2 toxicity and control of food hazards.

A highly sensitive of electrochemical biosensor based on new aptamer sequence for 17α-ethinylestradiol detection in water

The remarkable features of aptamer as biological agent in detecting various compound with high binding affinity could be useful in monitoring of 17α-ethinylestradiol (EE2). EE2 is considered as an endocrine disrupting compound due its ecotoxicity and has widespread in the aquatic ecosystem due to improper disposal. Hence, there is a pressing requirement for sensitive and selective detection of EE2. In this study, we developed a new electrochemical biosensor based on modified DNA aptamers and gold nanoparticles/poly(n-butyl acrylate-co-N-acryloxysuccinimide) microspheres (Au/P(nBA-NAS)). The DNA aptamers was truncated from a 76-mer sequence to 39-mer sequences via computational method, has showed improved binding affinity and stability for EE2. The biosensor was fabricated by attaching the aptamers to the Au/P(NBA-NAS) microspheres, which is provided a large surface area and enhanced electron transfer for the immobilization and signal amplification of the aptamers. The detection principle of the [Fe(CN)6]−3/−4 redox probe in differential pulse voltammetry (DPV) measurement by the presence of aptamer-EE2 complexes on the electrode surface. The biosensor exhibited a linear relationship between the current decrease and the EE2 concentration in the range of 1 × 10−6 M to 1 × 10−12 M, with a detection limit of 1.7 × 10−13 M. The biosensor exhibited excellent selectivity, repeatability, and recovery for detecting EE2 in real samples, with results that closely matched those obtained using the high-performance liquid chromatography (HPLC) technique. This study provided a promising alternative for developing aptamer-based sensors for onsite environmental monitoring.

Delivery of 5-fluorouracil for cancer therapy using aptamer-based nonlinear hybridization chain reaction

5-Fluorouracil (5-FU) is a conventional nucleotide analogue used for cancer treatment. However, its clinical application faces challenges such as low stability and non-specific toxicity. With the remarkable advancements in DNA nanotechnology, DNA-based self-assembled nanocarriers have emerged as powerful tools for delivering nucleotide drugs. In this study, we have designed a non-linear hybrid chain reaction involving a fuel strand with AS1411 aptamer sequence to construct a dendritic structure capable of carrying 5-FU. This structure specifically targets cancer cells with overexpressed nucleolin on their surface, allowing the 5-FU to exert its anticancer effects and achieve therapeutic outcomes. Furthermore, we have also investigated the mechanistic action of this drug delivery system, aiming to establish a novel therapeutic platform for 5-FU treatment.

A Tunable Threshold Colorimetric DNA Logic Gate for Intuitive Assessment of Chemical Contaminant Exceedance.

Current molecular logic gates are predominantly focused on the qualitative assessment of target presence, which has certain limitations in scenarios requiring quantitative assessment, such as chemical contaminant monitoring. To bridge this gap, we have developed a novel DNA logic gate featuring a tunable threshold, specifically tailored to the limits of contaminants. At the core of this logic gate is a DNA-gold nanoparticle (AuNP) hybrid film that incorporates aptamer sequences to selectively bind to acetamiprid (ACE) and atrazine (ATR). Upon interaction with these contaminants, the film degrades, releasing AuNPs that, in the presence of Hg2+, catalyze the oxidation of TMB, resulting in a visible blue coloration on test paper. This aptamer-enabled process effectively establishes an OR logic gate, with ACE and ATR as inputs and the appearance of blue color as the output. A key innovation of our system is its tunable input threshold. By adjusting the concentration of Hg2+, we can fine-tune the color mutation points to match the input threshold to predefined limits, such as Maximum Residue Limits (MRLs). This alignment allows semiquantitative assessment of contaminant levels, providing intuitive visual feedback of contaminant exceedance. Validation experiments with spiked samples confirm its accuracy and reliability by closely matching HPLC results. Therefore, our colorimetric DNA logic gate is emerging as a promising tool for easy and semiquantitative monitoring of chemical contaminants across diverse applications.

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.

Target-responsive triplex aptamer nanoswitch enables label-free and ultrasensitive detection of antibody in human serum via lighting-up RNA aptamer transcriptions

Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.

Highly catalytic sulfur-doped and bimetal-coordinated CoFe(CN)5NO nanoparticles coupled with PER/HCR amplification cascades for sensitive electrochemical aptamer luteinizing hormone assay

Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN)5NO (denoted as S–CoFe(CN)5NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S–CoFe(CN)5NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields.

Towards monitoring of critical illness via the detection of histones with extended gate field-effect transistor sensors

Extracellular histone proteins in the blood indicate a heightened risk of morbidity after trauma or in major illnesses such as sepsis. We present the development of an aptasensor for histone detection with an extended gate field-effect transistor (EGFET) configuration, which benefits from low power consumption, rapid response, and compatibility with miniaturized gold electrodes. Histones have a high isoelectric point and charge density, which cause them to physically adsorb to non-specific elements of the sensor that have available electrostatic charges. To combat this, the sensing surface is formed with a thiol-modified, high-affinity and histone-specific RNA aptamer sequence and by co-immobilizing with poly(ethylene glycol) methyl ether thiol (PEG) as a blocking agent. Surface plasmon resonance (SPR) is used to analyze aptamer and PEG immobilization strategies, confirm histone binding, and calculate kinetic binding constants. Through comparison of different blocking agents and time-dependent preparation, the ideal equilibrium dissociation constant (KD) is estimated to be below 200 pM, which is the upper range of extracellular histone concentrations in critically ill patients with high mortality. The EGFET sensitivity of the optimized aptasensor is 6.65 mV/decade concentration change for histone H4 with a physiologically relevant 5 pM limit of detection. Selectivity tests with 100 nM bovine serum albumin (BSA) demonstrate a signal response that is 13-fold smaller than for histones. This EGFET aptasensor platform is suitable for future point-of-care monitoring of histone levels in critically ill patients, thus permitting the early detection of increased risk and the need for more aggressive interventional measures to prevent mortality.

Exploring aptamers for targeted enrichment of X sperm in bovine: unraveling selective potential

Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.

CRISPR-Cas12a-mediated colorimetric aptasensor of netilmicin based on enzymes-assisted signal amplification and nanozyme employing a rationally truncated aptamer

Netilmicin (NET) is a typical veterinary antibiotic used in aquaculture and animal husbandry, but its excessive accumulation poses a serious threat to the ecological environment and human health. Therefore, a novel and highly sensitive Crispr-Cas12a-mediated colorimetric aptasensor was developed by combining the enzyme-assisted signal amplification strategy and magnetic NMOF-Pt nanozyme composite material (NPSM) for the visual detection of veterinary antibiotic residues in real samples. A high-affinity truncated aptamer (N-20) targeting NET was evolved from a parental aptamer sequence with the help of comprehensive recognition mechanism studies. Subsequently, N-20 was elaborately designed into the isothermal enzyme-assisted strand displacement amplification reaction. The amplified products (Act-DNAs) activated the trans-cleavage activity in the Crispr Cas12a system. The NMOF-Pt nanozyme was released from NPSM after cleaving the single chain of DNA (ssDNA). The quantity of released nanozyme was positively correlated with the amount of NET to quantitatively achieve the detection with the linear range and limits of detection (LOD) of about 0.003–30 ng/mL and 1.15 pg/mL, respectively. This method was successfully applied to detect NET in Lake water, milk and pork samples with satisfactory recoveries ranging from 89.9 % to 104.3 %. The constructed aptasensor demonstrated superior analytical performance and exhibited enormous application prospects in food supervision, environment monitoring, and medical laboratory science.

Research progress on the detection of foodborne pathogens based on aptamer recognition.

Foodborne diseases caused by bacterial contamination are a serious threat to food safety and human health. The classical plate culture method has the problems of long detection cycle, low sensitivity and specificity, and complicated operation, which cannot meet the growing demand for rapid quantitative detection of pathogenic bacteria. The frequent outbreak of foodborne diseases has put forward higher requirements for rapid and simple detection technology of foodborne pathogens. Aptamer is a kind of oligonucleotide fragment that can recognize targets with the advantages of high affinity and good specificity. The target can be range from proteins, small molecules, cells bacteria, and even viruses. Herein, the latest advances in sensitive and rapid detection of foodborne pathogens based on aptamer recognition was reviewed. Special attention has been paid to the obtained sequences of aptamers to various foodborne pathogens, the optimization of sequences, and the mechanism of aptamer recognition. Then, the research progress of biosensors for the detection of pathogenic bacteria based on aptamer recognition were summarized. Some challenges and prospects for the detection of foodborne pathogens based on aptamer recognition were prospected. In summary, with the further deepening of aptamer research and improvement of detection technology, aptamer-based recognition can meet the needs of rapid, sensitive, and accurate detection in practical applications.

Development of a Neuropeptide Y-Sensitive Implantable Microelectrode for Continuous Measurements.

In this work, we present the development of the first implantable aptamer-based platinum microelectrode for continuous measurement of a nonelectroactive molecule, neuropeptide Y (NPY). The aptamer immobilization was performed via conjugation chemistry and characterized using cyclic voltammetry before and after the surface modification. The redox label, methylene blue (MB), was attached at the end of the aptamer sequence and characterized using square wave voltammetry (SWV). NPY standard solutions in a three-electrode cell were used to test three aptamers in steady-state measurement using SWV for optimization. The aptamer with the best performance in the steady-state measurements was chosen, and continuous measurements were performed in a flow cell system using intermittent pulse amperometry. Dynamic measurements were compared against confounding and similar peptides such as pancreatic polypeptide and peptide YY, as well as somatostatin to determine the selectivity in the same modified microelectrode. Our Pt-microelectrode aptamer-based NPY biosensor provides signals 10 times higher for NPY compared to the confounding molecules. This proof-of-concept shows the first potential implantable microelectrode that is selectively sensitive to NPY concentration changes.

Aptamer biosensor design for the detection of endocrine-disrupting chemicals small organic molecules using novel bioinformatics methods

Endocrine-disrupting chemicals (EDCs) are substances that can disrupt the normal functioning of hormones.Using aptamers, which are biological recognition elements, biosensors can quickly and accurately detect EDCs in environmental samples. However, the elucidation of aptamer structures by conventional methods is highly challenging due to their complexity.This has led to the development of three-dimensional aptamer structures based on different models and techniques. To do this, we developed a way to predict the 3D structures of the SS DNA needed for this sequence by starting with an aptamer sequence that has biosensor properties specific to bisphenol-A (BPA), one of the chemicals found in water samples that can interfere with hormones. In addition, we will elucidate the intermolecular mechanisms and binding affinity between aptamers and endocrine disruptors using bioinformatics techniques such as molecular docking, molecular dynamics simulation, and binding energies. The outcomes of our study are to compare modeling programs and force fields to see how reliable they are and how well they agree with results found in the existing literature, to understand the intermolecular mechanisms and affinity of aptamer-based biosensors, and to find a new way to make aptamers that takes less time and costs less.

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.