Label-free Electrochemical Aptasensor Research Articles

A highly sensitive electrochemical aptasensor for detecting broad-spectrum estrogen molecules in clinical samples

The lack of sensitive and accurate monitoring methods for in vivo estrogen levels presents challenges for better prevention of estrogen-induced diseases. We have developed a label-free electrochemical aptasensor that demonstrates high sensitivity and selectivity for the broad-spectrum detection of estrogen molecules. This biosensor uses gold nanoparticles for electrochemical signal amplification and aptamers for broad-spectrum target recognition, enabling precise detection of trace amounts of estrogen in serum samples. The aptasensor demonstrates high sensitivity in estrogen detection, with a linear detection range of 0.01–1 nM and a minimum detection limit of 3 pM. It also exhibits excellent selectivity and interference resistance, with a detection error of less than 19 %, even in the presence of high concentrations of other biological substances. Additionally, molecular dynamics simulations were performed to provide insights into the molecular mechanism of aptamer broad-spectrum recognition and construction principles underlying the sensor. We anticipate that this aptasensor will serve as a robust, convenient, and cost-effective detection method, offering a valuable solution for the prevention of estrogen-induced diseases.

Portable label-free electrochemical aptasensor for sensitive detection of paraquat herbicide mediated by oxygen reduction reaction

Paraquat (PQ) is a highly toxic herbicide that has been prohibited in almost 70 countries, but remains in use worldwide. Thus, routine on-site PQ monitoring is a key mechanism to ensure safety and efficiently enforce regulations. Herein, a label-free portable electrochemical aptasensor for the detection of PQ was developed by utilizing aptamer designed to specifically recognize PQ. The aptasensor employs square-wave voltammetry (SWV) to quantify PQ binding on the aptamer-functionalized electrode surface by tracking the downstream oxygen reduction reaction. It provided a detection range spanning from 0.01 to 100.0 μg mL−1 PQ with a limit of detection (LOD) of 8.9 ng mL−1. Validation against spiked tap water, pomegranate juice, and orange juice revealed recovery rate performances of 75 %–130 %. The aptasensor demonstrates promising feasibility for PQ detection in real-world applications, offering remarkable portability and operational simplicity. Notably, it can operate without supplementary redox agents, requiring only sample incubation and subsequent washing steps.

Catalytic hairpin assembly-driven DNA walker to develop a label-free electrochemical aptasensor for antibiotic detection.

Anelectrochemical aptasensor was developed by utilizing a DNA walker driven by catalytic hairpin assembly (CHA) with kanamycin as the model analyte. Kanamycin bound to the aptamer, causesthe release of DNA walker, triggers the CHA reaction, leadsto the cyclic movement of the walker's long arm, and resultsin cascade amplification of thesignal. The guanine-rich sequences of the double-stranded products produced by CHA were folded to form G-quadruplex structures, with electrochemical active molecules Hemin embedded, formsG-quadruplex/Hemin complexes in situ on the electrode surface, thereby achieving sensitive, efficient, and label-free detection of kanamycin with a limit of detection (LOD) of 0.27pM (S/N = 3). Meaningfully, the aptasensor demonstrated high sensitivity and reliability in the detection of kanamycin in milk and livestock wastewater samples, suggesting that it has great potential for application in detecting antibiotics in food products and water samples from the environment.

Electrochemical Aptasensor with Antifouling Properties for Label-Free Detection of Oxytetracycline.

Oxytetracycline (OTC) is a widely employed antibiotic in veterinary treatment and in the prevention of infections, potentially leaving residues in animal-derived food products, such as milk, that are consumed by humans. Given the detrimental effects of prolonged human exposure to antibiotics, it has become imperative to develop precise and sensitive methods for monitoring the presence of OTC in food. Herein, we describe the development and results of a preliminary label-free electrochemical aptasensor with antifouling properties designed to detect OTC in milk samples. The sensor was realized by modifying a gold screen-printed electrode with α-lipoic acid-NHS and an amine-terminated aptamer. Different electrochemical techniques were used to study the steps of the fabrication process and to quantify OTC in the presence of the Fe(CN)64-/Fe(CN)63- redox couple The detectable range of concentrations satisfy the maximum residue limits set by the European Union, with an limit of detection (LOD) of 14 ng/mL in phosphate buffer (BP) and 10 ng/mL in the milk matrix, and a dynamic range of up to 500 ng/mL This study is a steppingstone towards the implementation of a sensitive monitoring method for OTC in dairy products.

Design of a facile and highly sensitive electrochemical impedimetric aptasensor for detection of 17β-estradiol

In this work, a facile, rapid and label-free electrochemical (EC) impedimetric aptasensor was developed for detecting 17β-estradiol (E2) based on gold particles (Au NPs) signal amplification. The aptamer was used as the recognition and capture unit to label on the surface of Au NPs. The anti-E2 aptamer labelled Au NPs (apt-Au NPs) were then anchored on the complementary DNA-immobilized gold electrode surface by hybridization. Owing to the large surface, good biocompatibility and high conductivity, Au NPs can not only provide a wonderful microenvironment for aptamer, but also play a signal amplification role, improve analytical performance of the EC sensing platform. Due to the high affinity and specificity of aptamer to E2, the formed aptamer-E2 complexes with poor conductivity increase the electron transfer resistance (Ret). Quantitative analysis for E2 was performed based on the correlation between the change of the Ret and E2 concentrations. The developed sensor exhibited excellent analytical performance with high sensitivity, selectivity and good stability for the detection of E2. The detection limit for E2 is 0.053 pM, with a linear response ranging 0.1–200 pM. Meanwhile, its application in real environmental samples was evaluated and satisfying results were achieved.

Selection of DNA aptamer for label-free electrochemical aptasensor for the detection of rubella virus

The Rubella virus (RUBV) is a highly contagious pathogen classified within the rubivirus genus, primarily infecting humans and transmitted via airborne routes. RUBV infection generally manifests as a mild illness reminiscent of measles. However, when affecting pregnant women, it can lead to a severe condition known as congenital rubella syndrome (CRS). Rubella infection could be also associated with joint pain, arthritis, and neurological disorders. Determination of Rubella immunity and diagnosis conventionally involve the Hemagglutination Inhibition (HI) test or the Enzyme-Linked Immunosorbent Assay (ELISA). In this study, we describe the selection and characterization of specific aptamers targeting the Rubella virus by using the process of Systematic Evolution of Ligands by EXponantial enrichment (SELEX). The Binding affinity studies have shown that the two aptamers; R-7 and R-5 display the lowest dissociation constants (Kd) of 6.58 nM and 19.05 nM, respectively. Then, R-7 aptamer was modified with a thiol group to enable its immobilization on screen-printed gold electrodes for the Rubella virus aptasensing. The label-free electrochemical detection was achieved using square wave voltammetry (SWV). The designed aptasensor has shown an excellent performance in detecting the Rubella virus within the range of 0.0005 ng/ml to 1000 ng/ml antigen and a limit of detection (LOD) of 0.00015 ng/ml. Selectivity studies were also performed against other viral antigens and serum proteins. Finally, the biosensor applicability was successfully demonstrated in spiked serum samples.

Catalytic hairpin-assembled and DNAzyme-driven DNA walker integrated hybridization chain reaction to construct a label-free electrochemical aptasensor for the detection of acetamiprid

As a new type of nicotine pesticide, the excessive residue of acetamiprid (ACE) in food and the environment seriously poses human health. Hence, this article developed an electrochemical aptasensor for detecting ACE based on a cascaded amplification circuit composed of catalytic hairpin-assembled and DNAzyme-driven DNA walker and hybridization chain reaction. Through the target triggered catalytic hairpin assembly (CHA), the free travelling strand of DNA walker and intact catalytic core of DNAzyme were formed, and with the assistance of Mg2+, DNAzyme was activated, driving the travelling strand to walk autonomously, repeatedly cutting hairpin DNA substrates on the electrode surface, and obtaining abundant DNA fragments. These fragments contained the initiators of hybridization chain reaction (HCR) could trigger HCR and produce the long double-strand DNA (dsDNA) for loading methylene blue (MB). Owing to the fact that Ce(Ⅲ, Ⅳ)-MOF material with electrocatalytic activity, the electrochemical reduction of MB could be effectively catalyzed, obtain significantly enhanced current signals, and ultimately achieve label-free and sensitive detection of ACE, with a detection limit of 16.38 fM (S/N = 3). Moreover, the aptasensor demonstrated excellent selectivity, stability, and reproducibility, and exhibited good reliability for detecting ACE in the vegetable soil and tomato samples, providing a new idea for the detection of pesticide residues in the fields of food and environment.

Label-free electrochemical aptasensor for the detection of HepG2 hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common liver malignancy and is characterized by increasing incidence and high mortality rates. Current methods for the screening and diagnosis of HCC exhibit inherent limitations, highlighting the ever-growing need for the development of new methods for the early diagnosis of HCC. The aim of this work was to develop a novel electrochemical aptasensor for the detection of HepG2 cells, a type of circulating tumor cells that can be used as biomarkers for the early detection of HCC. A carbon screen-printed electrode was functionalized with a composite suspension containing graphene oxide, chitosan, and polyaniline nanoparticles to increase the electrode surface and provide anchoring sites for the HepG2 cell-specific aptamer. The aptamer was immobilized on the surface of the functionalized electrode using multipulse amperometry, an innovative technique that significantly reduces the time required for aptamer immobilization. The innovative platform was successfully employed for the first time for the amplification-free detection of HepG2 cells in a linear range from 10 to 200,000 cells/mL, with a limit of detection of 10 cells/mL. The platform demonstrated high selectivity and stability and was successfully used for the detection of HepG2 cells in spiked human serum samples with excellent recoveries.Graphical abstract

Label-free electrochemical aptasensor for ultrasensitive lead ion detection based on flower-like AuNPs@MoS2 and core-shell Pt@Pd bimetallic nanoparticles.

A signal-amplified platform was designed to construct a label-free electrochemical aptasensor for lead ions (Pb2+) assay. First, flower-like molybdenum disulfide-supported AuNPs (AuNPs@MoS2) nanocomposites were synthesized and used as substrates for modifying the electrode. The AuNPs@MoS2 material possessed large surface area and superior biocompatibility, which was beneficial to improve the loading amount of the complementary DNA (cDNA) and amplified the response signal. Importantly, the prepared core-shell Pt@Pd bimetallic nanoparticles (Pt@PdNPs) were used to conjugate with redox marker thionine (Thi) and aptamer (Apt) for further signal amplification; the obtained signal probes (Thi-Pt@PdNPs-Apt) were connected by the cDNA assembled on the electrode through DNA hybridization. Differential pulse voltammetry was performed to monitor the signal of Thi. After incubating of aptasensor with Pb2+, the specific recognition of Pb2+ and Apt resulted in the dissociation of aptamer-cDNA complex, thereby the Thi-Pt@PdNPs-Apt separated from the electrode surface and decreased current response was obtained. The prepared electrochemical sensor exhibited linear response to Pb2+ in the range 5.0 × 10-4-100 nM and a detection limit of 1.0 × 10-4nM was achieved. The sensor was applied to the determination ofPb2+ in actual sample with high sensitivity and accuracy, demonstrating potential applications in heavy metal monitoring.

Selection of a new aptamer targeting amoxicillin for utilization in a label-free electrochemical biosensor

Pharmaceutical pollution has received considerable attention because of the harmful effects of pharmaceutical compounds on human health, even in trace amounts. Amoxicillin is one of the frequently used antibiotics that was included in the list of emerging water pollutants. Therefore, a highly selective and rapid technique for amoxicillin detection is required. In this work, a new aptamer was selected for amoxicillin and utilized for the development of a label-free electrochemical aptasensor. Aptamer selection was performed using the systematic evolution of ligands by exponential enrichment. The selected aptamer showed good specificity against other antibiotics, including the structurally related antibiotics: ampicillin and ciprofloxacin. Among the selected aptamers, Amx3 exhibited the lowest dissociation constant value of 112.9 nM. An aptasensor was developed by immobilization of thiolated Amx3 aptamer onto gold screen–printed electrodes via self-assembly, which was characterized using cyclic voltammetry and electrochemical impedance spectroscopy. The detection was realized by monitoring the change in the differential pulse voltammetry peak current in the ferro/ferricyanide redox couple upon binding of the aptasensor to amoxicillin. The aptasensor showed very good sensitivity with an ultralow limit of detection of 0.097 nM. When the aptasensor was tested using actual spiked milk samples, excellent recovery percentages were observed. The label-free electrochemical aptasensor developed herein is a promising tool for the selective and sensitive detection of amoxicillin in environmental samples.

Ultrasensitive label-free electrochemical aptasensor for Pb2+ detection exploiting Exo III amplification and AgPt/GO nanocomposite-enhanced transduction

Lead ion pollution has become a serious public health concern worldwide. Therefore, sensitive detection of Pb2+ is critical to control lead pollution, assess risks, and safeguard the health of vulnerable populations. This study reports a highly sensitive labelling-free electrochemical aptasensor for Pb2+ detection. The aptasensor employs silver-platinum nanoparticles/graphene oxide (AgPt/GO) and Exonuclease III (Exo III) for signal amplification. GO provides high surface area and conductivity for immobilizing AgPt NPs, facilitating the immobilization of aptamer (Apt) probes on the electrode surface. Exo III enzymatically cleaves DNA strands on the electrode surface, releasing DNA segments to amplify the signal further. The synergistic amplification by AgPt/GO and ExoIII enables an extremely wide linear detection range of 0.05 pM–5 nM for Pb2+, with a low detection limit of 0.019 pM. Additionally, the G-quadruplex structure ensures excellent selectivity for Pb2+ detection, resulting in high reproducibility and stability of the aptasensor. The aptasensor was successfully applied to detect spiked Pb2+ in tap water samples, achieving recovery rates ranging from 96 to 108.4 %. By integrating nanomaterials, aptamers and enzymatic amplification, the aptasensor facilitates highly sensitive and selective detection of Pb2+, demonstrating potential for practical applications in environmental monitoring.

Design of label-free electrochemical aptasensors for enrofloxacin detection in mutton

Design of label-free electrochemical aptasensors for enrofloxacin detection in mutton

Label-free electrochemical aptasensor for detection of Acinetobacter baumannii: Unveiling the kinetic behavior of reduced graphene oxide v/s graphene oxide

In this study, we introduce an electrochemical aptasensor for the sensitive detection of Acinetobacter baumannii. This method, based on impedance and current analysis, represents a first-of-its kind platform in the field of A. baumannii detection. We employed a screen-printed carbon electrode modified with both graphene oxide (GO) and reduced graphene oxide (rGO) as the immobilization platform, and the bacteria specific aptamer was employed as the biorecognition element. The aptamer selectively interacts with A. baumannii, causing a decrease in the peak current of the redox complex because the binding biomolecules on the electrode surface hinder the electron transfer of the redox probe. This initial point-of-care-compatible rGO electrochemical aptasensor demonstrated an exceptional sensing performance with an impressive limit of detection of 10 CFU/mL for detecting A. baumannii. This enhanced sensitivity can be attributed to several key factors including structural defects during reduction, increased surface area and improved electron transfer kinetics. The findings of this study highlight the potential of the developed aptasensor as a rapid and reliable detection platform for various pathogenic diseases. By further ensuring seamless integration into clinical practice, these label-free graphene-based aptasensors, with their π-π interactions and other favorable attributes, hold great promise for sensitive and specific pathogen detection, thereby contributing to advancements in healthcare diagnostics.

Enhanced sensitivity in electrochemical detection of ochratoxin A within food samples using ferrocene- and aptamer-tethered gold nanoparticles on disposable electrodes.

Ensuring food security is crucial for public health, and the presence of mycotoxins, produced by fungi in improperly stored processed or unprocessed food, poses a significant threat. This research introduces a novel approach - a disposable aptasensing platform designed for the detection of ochratoxin A (OTA). The platform employs gold-nanostructured screen-printed carbon electrodes functionalized with a ferrocene derivative, serving as an integrated faradaic transducing system, and an anti-OTA aptamer as a bioreceptor site. Detection relies on the ferrocene electrochemical signal changes induced by the aptamer folding in the presence of the target molecule. Remarkably sensitive, the platform detects OTA within the range of 0.5 to 70 ng mL-1 and a detection limit of 11 pg mL-1. This limit is approximately 200 times below the levels stipulated by the European Commission for agricultural commodities. Notably, the sensing device exhibits efficacy in detecting OTA in complex media, such as roasted coffee beans and wine, without the need for sample pretreatment, yielding accurate recoveries. Furthermore, while label-free electrochemical aptasensors have proliferated, this study addresses a gap in understanding the binding mechanisms of some aptasensors. To enhance the experimental findings, a theoretical study was conducted to underscore the specificity of the anti-OTA aptamer as a donor for OTA detection. The molecular docking technique was employed to unveil the key binding region of the aptamer, providing valuable insights into the aptasensor specificity.

3D nanocake-like Au-MXene/Au pallet structure-based label-free electrochemical aptasensor for paraquat determination.

3D nanocake-like Au-MXene and Au pallet (Au-MXene/AuP) nanocomposite-modified screen-printed carbon electrodes (SPCEs) were utilized to construct an ultrasensitive label-free electrochemical aptasensor through a self-assembly procedure for trace paraquat (PQ) residue detection. Benefiting from the excellent electrochemical (EC) performances (e.g., high conductivity and large surface area) of Au-MXene nanocomposites and AuP substrate, the developed Apt/Au-MXene/AuP/SPCE-based EC aptasensor displayed excellent specificity and anti-interference ability, good repeatability, and stability. A linear relationship between the log value of the change in current intensity [lg (ΔI)] and the log value of the concentration of PQ [lg (CPQ)] was obtained in the range 0.05-1000 ng/mL. The limit of detection was 0.028 ng/mL, and thesensitivity was 255.5 μA/(μM·cm2). Practical applications in malt and mint samples confirmed the accuracy of the EC aptasensor in complex matrices for PQ detection, providing a universal analytical tool for othertrace pesticides in differentfood samples by simply replacing the corresponding aptamers.

A label-free electrochemical aptasensor based on Ti3C2Tx-Ag/Au nanoparticles as a signal amplification strategy for CRP detection

C-reactive protein (CRP) is an essential biomarker that is associated to acute inflammation and infectious illnesses. This study developed a label-free electrochemical aptasensor utilizing Ti3C2Tx-silver/gold nanoparticles (Ti3C2Tx-Ag/Au NPs) for the first time for CRP detection. Based on the unique capacity of Ti3C2Tx of self-adsorbing and reducing silver ions (Ag+), Ag signal probes with strong redox activity and good electrical conductivity were synthesized. Electrodeposition of Au NPs on the electrode surface modified with Ti3C2Tx-Ag, not merely improved the adsorption capacity of the immobilized aptamer and prevented Ag loss; it also collaborated with Ti3C2Tx-Ag to create a dual-amplified signal strategy. Aptamer molecules bind to Au NPs through the Au-S bond for precise capture of CRP. The detection was performed by differential pulse voltammetry (DPV), the current was proportional to the logarithm of CRP antigen, and the linear range of CRP detection was 0.1–200 ng mL−1 with a limit of detection (LOD) 41 pg mL−1. Simultaneously, the label-free aptasensor presented superior detection selectivity, reproducibility, and stability. This study provided an ideal electrochemical biosensor for determining CRP in serum, with an excellent prospect for clinical diagnostic applications at the point of care testing (POCT).

Label-Free Homogeneous Electrochemical Aptasensor Based on Size Exclusion/Charge-Selective Permeability of Nanochannel Arrays and 2D Nanorecognitive Probe for Sensitive Detection of Alpha-Fetoprotein.

The labeling-free and immobilization-free homogeneous aptamer sensor offers advantages including simple operation, low cost, and high sensitivity, demonstrating great potential in rapid detection of tumor biomarkers in biological samples. In this work, a labeling-free and immobilization-free homogeneous aptamer sensor was conveniently fabricated by combining size exclusion and charge-selective penetration of a nanochannel-modified electrode and two-dimensional (2D) nanorecognition probe which can realize selective and highly sensitive detection of alpha-fetoprotein (AFP) in serum. Vertically ordered mesoporous silica film (VMSF) with ultra-small, uniform, and vertically aligned nanochannels was easily grown on the simple, low-cost, and disposable indium tin oxide (ITO) electrode. Through π-π interaction and electrostatic force, the AFP aptamer (Apt) and electrochemical probe, tris(bipyridine)ruthenium(II) (Ru(bpy)32+), were coloaded onto graphene oxide (GO) through simple incubation, forming a 2D nanoscale recognition probe (Ru(bpy)32+/Apt@GO). Owing to the size exclusion effect of VMSF towards the 2D nanoscale probe, the electrochemical signal of Ru(bpy)32+/Apt@GO could not be detected. In the presence of AFP, the specific binding of AFP to the aptamer causes the dissociation of the aptamer and Ru(bpy)32+ from GO, resulting in their presence in the solution. The efficient electrostatic enrichment towards Ru(bpy)32+ by negatively charged VMSF allows for high electrochemical signals of free Ru(bpy)32+ in the solution. Linear determination of AFP ranged from 1 pg/mL to 1000 ng/mL and could be obtained with a low limit of detection (LOD, 0.8 pg/mL). The high specificity of the adapter endowed the constructed sensor with high selectivity. The fabricated probe can be applied in direct determination of AFP in serum.

Rapid and label-free electrochemical aptasensor based on a palladium nanoparticles/titanium carbide/polyethyleneimine functionalized nitrogen-doped carbon nanotubes composite for amplified detection of streptomycin

A novel electrochemical aptasensor for the determination of streptomycin (STR) was constructed in this work. We prepared a palladium nanoparticles/titanium carbide/polyethyleneimine–functionalized nitrogen-doped carbon nanotube (Pd@Ti3C2-PEI-NCNTs) composite via hydrothermal-assisted formic acid reduction and employed it as a substrate material for anchoring a NH2-functionalized aptamer (Apt) to the surface of an electrode through the Pd-N bond. The Pd@Ti3C2-PEI-NCNTs composite increased the electrochemically active surface area and conductivity of the electrode, achieving substantial signal amplification (1.87 times). The constructed aptasensor demonstrated accurate STR detection via specific recognition between the Apt and STR, with a detection range and limit of 0.01–700 and 0.003 nmol/L, respectively. The aptasensor also exhibited excellent selectivity, stability, reproducibility, practicability and shorter assay times (60 min). Furthermore, molecular docking and molecular dynamic also elucidated the binding mechanism between the aptamer and STR molecules. Overall, this method is expected to be a helpful tool for detecting STR in food.

Few Layer Ti3C2 MXene-Based Label-Free Aptasensor for Ultrasensitive Determination of Chloramphenicol in Milk.

Quantitative detection of veterinary drug residues in animal-derived food is of great significance. In this work, a simple and label-free electrochemical aptasensor for the highly sensitive detection of chloramphenicol (CAP) in milk was successfully developed based on a new biosensing method, where the single- or few-layer Ti3C2 MXene nanosheets functionalized via the specific aptamer by self-assembly were used as electrode modifiers for a glassy carbon electrode (aptamer/Ti3C2 MXene/GCE). Differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscopy (SEM), atomic force microscope (AFM), and so on were utilized for electrochemical and morphological characterization. Under the optimized conditions, the constructed aptasensor exhibited excellent performance with a wider linearity to CAP in the range from 10 fM to 1 μM and a low detection limit of 1 fM. Aptamer/Ti3C2 MXene/GCE demonstrated remarkable selectivity over other potentially interfering antibiotics, as well as exceptional reproducibility and stability. In addition, the aptasensor was successfully applied to determine CAP in milk with acceptable recovery values of 96.13% to 108.15% and relative standard deviations below 9%. Therefore, the proposed electrochemical aptasensor is an excellent alternative for determining CAP in food samples.

Truncated affinity-improved aptamer for selective and sensitive detection of streptomycin in dairy products with label-free electrochemical aptasensor

The overuse of streptomycin (STR) can result in their residues in food, posing serious side effects on human health. This work aims to develop a novel and accurate electrochemical aptasensor for the detection of STR in dairy products. Owing to the interaction of a portion of the aptamer with its target, we have rationally truncated an already existing 79-mer STR aptamer sequence, obtaining a smaller size, lower synthesis cost, and higher affinity truncated aptamer (S03 aptamer). The favorable affinity of S03 aptamer to STR was further verified. Then, an electrochemical aptasensor was established using S03 as a recognition molecule and [email protected] carbon nanotube/Au nanoparticles ([email protected]/AuNPs) as support matrix for the detection of STR. The detection limit of the S03 aptamer was lower than that of the full-length. It is preliminary proved that S03 truncated aptamer can be used as a recognition element for STR detection.