An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ο-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3′ terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100 fM to 1 nM with a low detection limit of 20.3 fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future.     

Electrochemical Aptasensor Based on Polycarboxylic Macrocycle Modified with Neutral Red for Aflatoxin B1 Detection

Novel electrochemical aptasensors based on glassy carbon electrodes modified with electropolymerized Neutral red and polycarboxylated macrocyclic ligands onto which the DNA aptamers were covalently attached have been developed for detection of Aflatoxin B1 (AFB1). The interaction with an analyte resulted in the decrease of the cathodic peak current of the probe measured by CV and in the increase of the electron transfer resistance determined by EIS. The limit of detection was found to be 0.1 nM for CV and 0.05 nM for EIS methods, respectively. The aptasensor makes it possible to detect AFB1 in peanuts, cashew nuts, white wine and soy sauce with a recovery of 85–100 %.     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

Electrochemical Aptasensor for Zearalenone Based on DNA Assembly and Exonuclease III as Amplification Strategy

A sensitively electrochemical aptasensor was developed to detect zearalenone, utilizing DNA assembly based on hybridization chain reaction to amplify the signal current and exonuclease III to reduce the background current. The linear range 5.0×10⁻⁵ ng/mL-50 ng/mL, and the limit of detection is 0.013 pg/mL. The fabricated aptasensor showed the high specificity toward aflatoxin B1 (AFB1), fumonisin B1 (FB1) and ochratoxin A (OTA), good repeatability and reproducibility. In addition, the average recoveries of spiked corn and beer samples were in the range of 89 % to 102 %. The established method is of great significance in the field of food safety detection.     

Microfluidic chip-based aptasensor for amplified electrochemical detection of human thrombin

In this paper, a microchip-based sandwich-type aptasensor is developed for the detection of human thrombin. The SH-aptamer/thrombin/alkaline phosphatase-functionalized aptamer (ALP-aptamer) system was constructed in the microfluidic channels. And the substrate solution containing 4-aminophenyl phosphate (p-APP) was introduced to the microchannels for the end-column electrochemical detection. The on-chip aptasensor has a broad linear response range of 1–100 pM with a detection limit of 1 pM, which shows high sensitivity and specificity. The system was then applied to detect thrombin in human serum sample. Therefore, the on-chip aptasensor has a great promise for detecting and screening ultratrace levels of biomarkers in the complex matrices.     

Label-free Electrochemical Aptasensor for the Determination of Serotonin

A label-free electrochemical aptasensor was fabricated for the determination of serotonin (5-HT) by the employment of specific binding between 5-HT and aptamer and directly electrochemical measurement of 5-HT oxidation. The comparison between two aptasensors and two strategies was studied. An electrochemical aptasensor fabricated using 57-mer aptamer was facilely used to determine 5-HT in the range from 1 μM to 100 μM with detection limit of 0.3 μM via one step recognition and detection. This work demonstrates that electrochemical method combing aptamer recognition and 5-HT oxidation provides an alternative and promising method for the determination of 5-HT with good selectivity and simplicity.     

基于Cu-TPA的电化学生物传感器对黄曲霉毒素B1的检测

利用对苯二甲酸铜(Cu-TPA)能产生强的电化学信号设计了一种灵敏的电化学生物传感器, 并将其用于测定黄曲霉毒素B1(AFB1). 信号探针中的Cu-TPA含有可产生电化学信号的Cu(Ⅱ), 当加入一定量的AFB1后, AFB1与探针中特定的适配体结合, 使信号探针脱落, 电化学信号降低. 根据电化学信号值的变化实现了对AFB1的检测. 在最佳条件下, 该传感器的检出限为4.2×10 ^-6 ng/mL(S/N=3), 线性范围为10 ^-5~10 ng/mL. 将该传感器用于啤酒中AFB1的检测, 回收率为95%~106%.     

Ultrasensitive one-step rapid detection of ochratoxin A by the folding-based electrochemical aptasensor

A one-step electrochemical aptasensor using the thiol- and methylene blue- (MB-) dual-labeled aptamer modified gold electrode for determination of ochratoxin A (OTA) was presented in this research. The aptamer against OTA was covalently immobilized on the surface of the electrode by the self-assembly effect and used as recognition probes for OTA detection by the binding induced folding of the aptamer. Under the optimal conditions, the developed electrochemical aptasensor demonstrated a wide linear range from 0.1 pg mL⁻¹ to 1000 pg mL⁻¹ with the limit of detection (LOD) of 0.095 pg mL⁻¹, which was an extraordinary sensitivity compared with other common methods for OTA detection. Moreover, as a practical application, this proposed electrochemical aptasensor was used to monitor the OTA level in red wine samples without any special pretreatment and with satisfactory results obtained. Study results showed that this electrochemical aptasensor could be a potential useful platform for on-site OTA measurement in real complex samples.     

基于石墨烯化学修饰电极的适体传感器

采用石墨烯(RGO)作载体,凝血酶适体(TBA)作探针,凝血酶为目标蛋白,电化学阻抗谱(EIS)为检测技术,建立了检测蛋白质的新方法。由于RGO可增大电极有效表面积并提高电极表面电子传输速率以及TBA的特异性识别能力,此方法具有较高的灵敏度和良好的选择性。采用本方法检测凝血酶的线性范围为0.3～10 fmol/L,检出限为0.26 fmol/L。本研究将RGO应用于电化学适体传感器,证实了RGO修饰电极在电化学适体传感器领域中潜在的应用价值。     

基于碳纳米管的高灵敏度免标记电化学核酸适体传感电极

以电活性钌化合物[Ru(NH₃)₆]³⁺为信号传感源,借助碳纳米管构建了高灵敏检测腺苷免标记电化学传感电极（BSA/Apt/CNTs/GC）. BSA/Apt/CNTs/GC电极在最佳实验条件下检测腺苷线性范围为5.0×10^-11 ~ 1.0×10^-7 mol·L^-1,检测下限为2.7×10^-11 mol·L^-1. 该传感电极有较高的灵敏度、良好的选择性、重现性和稳定性. 与传统标记型适体传感电极相比,其制作简便,也许还适用于其他小分子和蛋白质的检测,有一定的普适性.     

A signal amplification by QDs used for ferrocene-labeled sandwich aptasensor for determination of Hg2+ in water samples

In this paper, the design of a novel sandwich-type electrochemical aptasensor was reported for an ultrasensitive mercury ion (Hg²⁺) detection in water samples, which labeled with two-labeled aptamer (Apt) sequences. The used Apts were Apt1 and Apt2 as the capture and signal probe, respectively. The Apt1 probe was immobilized on the poly(4-aminobenzoic acid) (p-ABA) and quantum dots (QDs) film as the platform, as well as the Apt2 reporter was labeled with ferrocene. In the presence of Hg²⁺, the strong coordination complex has been formed between the specific thymine of the Apt1, Hg²⁺, as well as the thymine of the Apt as T–Hg²⁺–T adduct. The QDs and p-ABA were applied for increasing the conductivity of platform and suitable binding of the recognition elements. Under the optimized conditions, the constructed aptasensor illustrated either a wide linear relationship between the logarithm of Hg²⁺ concentration and current, from 0.05 to 100 nM and also an excellent low limit of detection of 0.01 nM. The quality of carefully choosing, an excellent stability and specificity sensitivity of the designed aptasensor, was investigated by spiked tap water samples as real sample. Moreover, the aptasensor exhibits the good reproducibility as well as has high selectivity for the other cations. The recoveries of the Hg²⁺ assay of the tap water samples were acquired satisfactorily which imply the generated aptasensor can use Hg²⁺ measurement in the real laboratories.

     

快速检测中药材中黄曲霉毒素B1的电化学生物传感器

结合新型纳米传感膜材料,以黄曲霉毒素Bl(AFBl)单克隆抗体为生物识别元件,通过考察AFB1与抗体之间的相互作用对测试底液中[Fe(CN)6]3-/[Fe(CN)6]4-([Fe(CN)6]3-/4-)氧化还原体系的影响,构建了一种可用于中药材中AFB1快速检测的电化学生物传感器.在最佳条件下,对AFB1浓度的线性响...     

An aptamer-based biosensing platform for highly sensitive detection of platelet-derived growth factor via enzyme-mediated direct electrochemistry

In this work, a new label-free electrochemical aptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growth factor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). For this proposed aptasensor, poly(diallyldimethylammonium chloride) (PDDA)-protected graphene-gold nanoparticles (P-Gra-GNPs) composite was firstly coated on electrode surface to form the interface with biocompatibility and huge surface area for the adsorption of GOD layer. Subsequently, gold nanoclusters (GNCs) were deposited on the surface of GOD to capture PDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to block the remaining active sites of the GNCs and avoid the nonspecific adsorption. With the direct electron transfer of double layer GOD membranes, the aptasensor showed excellent electrochemical response and the peak current decreased linearly with increasing logarithm of PDGF concentration from 0.005 nM to 60 nM with a relatively low limit of detection of 1.7 pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-term stability, which provided a new promising technique for aptamer-based protein detection.     

A highly selective and sensitive cocaine aptasensor based on covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support platform

Based on the conformational changes of the aptamer-functionalized gold nanoparticles (AuNPs) onto MWCNTs/IL/Chit nanocomposite as the support platform, we have developed a sensitive and selective electrochemical aptasensor for the detection of cocaine. The 5′-amine-3′-AuNP terminated aptamer is covalently attached to a MWCNTs/IL/Chit nanocomposite. The interaction of cocaine with the aptamer functionalized AuNP caused the aptamer to be folded and the AuNPs with negative charge at the end of the aptamer came to the near of electrode surface therefore, the electron transfer between ferricyanide (K₃Fe(CN)₆) as redox probe and electrode surface was inhibited. A decreased current of (K₃Fe(CN)₆) was monitored by differential pulse voltammetry technique. In an optimized condition the calibration curve for cocaine concentration was linear up to 11 μM with detection limit (signal-to-noise ratio of 3) of 100 pM. To test the selectivity of the prepared aptasensor sensing platform applicability, some analgesic drugs as the interferes were examined. The potential of the aptasensor was successfully applied for measuring cocaine concentration in human blood serum. Based on our experiments it can be said that the present method is absolutely beneficial in developing other electrochemical aptasensor.     

Fabrication of highly sensitive and selective nanocomposite film based on CuNPs/fullerene-C60/MWCNTs: An electrochemical nanosensor for trace recognition of paracetamol

Designing an electrochemical sensor for versatile clinical applications is a sophisticated task and how dedicatedly functionalized composite materials can perform on this stage is a challenge for today and tomorrow's Nanoscience and Nanotechnology. In the present work, we demonstrate a new strategy for the development of novel electrochemical sensor based on catalytic nanocomposite film. Fullerene-C₆₀ and multi-walled carbon nanotubes (MWCNTs) were dropped on the pre-treated carbon paste electrode (CPE) and copper nanoparticles (CuNPs) electrochemically deposited on the modified CPE to form nanocomposite film of CuNPs/C₆₀/MWCNTs/CPE. In this work, an electrochemical method based on square wave voltammetry (SWV) employing CuNPs/C₆₀/MWCNTs/CPE has been presented for the recognition and determination of paracetamol (PT). Developed electrochemical sensor was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronocoulometry. The composite film made the fabricated sensor to display high sensitivity and good selectivity for PT detection. The influence of the optimization parameters such as pH, accumulation time, deposition potential, scan rate and effect of loading of composite mixture of C₆₀-MWCNTs and CuNPs on the electrochemical performance of the sensor were evaluated. A linear range from 4.0 × 10⁻⁹ to 4.0 × 10⁻⁷ M for PT detection was obtained with a detection limit of 7.3 × 10⁻¹¹ M. The fabricated sensor was successfully applied to the detection of PT in biological samples with good recovery ranging from 99.21 to 103%.     

Direct detection of OTA by impedimetric aptasensor based on modified polypyrrole-dendrimers

Ochratoxin A (OTA) is a carcinogenic mycotoxin that contaminates food such as cereals, wine and beer; therefore it represents a risk for human health. Consequently, the allowed concentration of OTA in food is regulated by governmental organizations and its detection is of major agronomical interest. In the current study we report the development of an electrochemical aptasensor able to directly detect trace OTA without any amplification procedure. This aptasensor was constructed by coating the surface of a gold electrode with a film layer of modified polypyrrole (PPy), which was thereafter covalently bound to polyamidoamine dendrimers of the fourth generation (PAMAM G4). Finally, DNA aptamers that specifically binds OTA were covalently bound to the PAMAM G4 providing the aptasensor, which was characterized by using both Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) techniques. The study of OTA detection by the constructed electrochemical aptasensor was performed using Electrochemical Impedance Spectroscopy (EIS) and revealed that the presence of OTA led to the modification of the electrical properties of the PPy layer. These modifications could be assigned to conformational changes in the folding of the aptamers upon specific binding of OTA. The aptasensor had a dynamic range of up to 5 μg L⁻¹ of OTA and a detection limit of 2 ng L⁻¹ of OTA, which is below the OTA concentration allowed in food by the European regulations. The efficient detection of OTA by this electrochemical aptasensor provides an unforeseen platform that could be used for the detection of various small molecules through specific aptamer association.     

Sensitive Electrochemical Aptasensor for Thrombin Detection with Platinum Nanoparticles,Blocking Reagent‐Horseradish Peroxidase and Graphene Oxide as Enhancers

A highly sensitive thrombin electrochemical aptasensor with Pt nanoparticles, blocking reagent‐horseradish peroxidase (HRP) and inert graphene oxide (GO) as enhancers was successfully fabricated. Firstly, Pt nanoparticles with high surface to volume ratio could increase the amount of the immobilized redox probe hexacyanoferrate nanoparticles (NiHCFNPs) and effectively enhance the electron transfer. Secondly, HRP and Pt nanoparticles with high catalytic activity extremely amplify the electrochemical signal of NiHCFNPs toward H₂O₂. Lastly, inert graphene oxide (GO) labeled TBA could be used for enlarging the steric hindrance of thrombin. As a result, the aptasensor showed a high sensitivity with a detection limit of 500 fM.     

MXene–AuNP-Based Electrochemical Aptasensor for Ultra-Sensitive Detection of Chloramphenicol in Honey

A simple and label-free electrochemical aptasensor was developed for ultra-sensitive determination of chloramphenicol (CAP) based on a 2D transition of metal carbides (MXene) loaded with gold nanoparticles (AuNPs). The embedded AuNPs not only inhibit the aggregation of MXene sheets, but also improve the quantity of active sites and electronic conductivity. The aptamers (Apts) were able to immobilize on the MXene–AuNP modified electrode surface through Au–S interaction. Upon specifically binding with CAP with high affinity, the CAP–Apt complexes produced low conductivity on the aptasensor surface, leading to a decreased electrochemical signal. The resulting current change was quantitatively correlated with CAP concentration. Under optimized experimental conditions, the constructed aptasensor exhibited a good linear relationship within a wide range of 0.0001–10 nM and with a low detection limit of 0.03 pM for CAP. Moreover, the developed aptasensor has been applied to the determination of CAP concentration in honey samples with satisfactory results.     

Detection of Allergenic Lysozyme during Winemaking with an Electrochemical Aptasensor

In this work we investigate the performance of a simple, disposable electrochemical aptasensor for lysozyme and its usefulness for monitoring the allergen risk along wine production. The sensor relies on screen‐printed gold electrodes modified with gold nanoparticles as the electrochemical transducer, with detection by cyclic voltammetry. This simple method is characterized by a detection limit of 0.32 μg.mL^?1 lysozyme and a linear range of 1–10 μg.mL^?1, being appropriate for the analysis of lysozyme‐treated wines. Several white wines where sulphur dioxide was partially replaced by lysozyme were produced and analyzed with the aptasensor at critical stages during wine production. The results obtained with the aptasensor were moreover compared with those recorded in parallel by a standard method, high performance liquid chromatography (HPLC). The specific advantages brought by the use of nanomaterial and the limitations of the sensor are discussed. The sensor allowed evaluating the effect of various technological steps along wine production on the content of lysozyme.     

A Hairpin Electrochemical Aptasensor for Sensitive and Specific Detection of Thrombin Based on Homogenous Target Recognition

An electrochemical aptasensor was developed for sensitive and specific detection of thrombin by combining homogenous recognition strategy and gold nanoparticles (AuNPs) amplification. Streptavidin‐alkaline phosphatase was used as reporter molecule. Compared with the traditional hairpin aptasensor monitoring the distance of the redox molecule from the electrode surface, the proposed aptasensor successfully overcome the limitations of distance and improved the stability and high affinity of the aptamer hairpin through homogenous recognition, which enhanced the sensitivity and selectivity of the sensors effectively. Additionally, AuNPs were employed to increase the active area and conductivity of the electrode, thus, improving the sensitivity of the aptasensor. As a result, the designed thrombin detection sensor obtained a lower detection limit of 0.52 pM in buffer and 6.9 pM in blood serum.