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.     

An aptamer-based biosensor for sensitive thrombin detection

A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using quantum dots as electrochemical label. CdSe quantum dots were labeled to the secondary aptamer, which were determined by the square wave stripping voltammetric analysis after dissolution with nitric acid. The aptasensor has a lower detection limit at 1 pM, while the sample consumption is reduced to 5 μl. The proposed approach shows high selectivity and minimizes the nonspecific adsorption, so that it was used for the detection of target protein in the human serum sample. Such an aptamer-based biosensor provides a promising strategy for screening biomarkers at ultratrace levels in the complex matrices.     

Electrogenerated chemiluminescence aptamer-based biosensor for the determination of cocaine

A novel electrogenerated chemiluminescence aptamer-based (ECL-AB) biosensor for the determination of a small molecule drug is designed employing cocaine-binding aptamer as molecular recognition element for cocaine as a model analyte and ruthenium complex served as an ECL label. A 5′-terminal cocaine-binding aptamer with the ECL label at 3′-terminal of the aptamer was utilized as an ECL probe. The ECL-AB biosensors were fabricated by immobilizing the ECL probe onto a gold electrode surface via thiol-Au interactions. An enhanced ECL signal is generated upon recognition of the target cocaine, attributed to a change in the conformation of the ECL probe from random coil-like configuration on the probe-modified film to three-way junction structure, in close proximity to the sensor interface. The integrated ECL intensity versus the concentration of cocaine was linear in the range from 5.0 × 10⁻⁹ to 3.0 × 10⁻⁷ M. The detection limit was 1.0 × 10⁻⁹ M. This work demonstrates that the combination of a highly binding aptamer to analyte with a highly sensitive ECL technique to design ECL-AB biosensor is a great promising approach for the determination of small molecule drugs.     

Electrochemical Aptasensor Based on ZnO Modified Gold Electrode

We developed an electrochemical thrombin aptasensor based on ZnO nanorods functionalized by electrostatically adsorption of 30‐mer DNA aptamers. The sensor surface was characterized by AFM and SEM. The surface layer assembling was optimized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with ferricyanide ions as redox markers. The peak current of the ferricyanide and the charge transfer resistance gradually decreased with increasing concentration of thrombin in the range from 3 pM to 100 nM due to formation of aptamer‐thrombin complexes and slower diffusion of the marker ions through the surface layer. At optimal conditions, a limit of detection (LOD) of 3 pM for EIS measurements and 10 pM for CV response was calculated from the S/N=3.     

基于主客体竞争模式的凝血酶适配体传感器的研究

基于β-环糊精(β-CD)主客体竞争模式,构建了开关型凝血酶适配体电化学传感器.将末端修饰了二茂铁(Fc)的核酸适配体通过与β-CD的主客体识别固定在金电极表面,当凝血酶存在时,适配体由原来的直立线状构型变为"G-四链体",远离电极表面,适配体探针的氧化还原电流强度减小,即"Signal-off".利用此效应对凝血酶进行了灵敏检测,结果表明,在5.0×10-13~5.0×10-9 mol/L浓度范围内,凝血酶的浓度与电化学响应信号呈良好的线性关系,检出限为2.0×10-13 mol/L(3σ).与其它蛋白分子相比,本方法对凝血酶蛋白的检测具有高特异性.本传感器构建简单,再生性好,为生物血清样本中凝血酶的实时高效检测提供了方法.     

Noncovalent assembly of ferrocene on modified gold surfaces mediated by uracil–adenine base pairs

Redox-active ferrocene was assembled on gold surfaces through the hydrogen bonding interactions between adenine-substituted ferrocene and a uracil-terminated organothiol monolayer. The surface coverage of ferrocene Γ could be varied from ca. 4 × 10^? 11 to 2.0 × 10^? 10 mol cm^? 2 by diluting the thiol-modified uracil derivative with inert 1-octanethiol. A decrease in the apparent electron transfer rate constant for ferrocene, k_app, from ca. 50 to 10 s^? 1 was observed upon increasing Γ.     

基于铁氰化镍的非标记型核酸适配体电化学传感器检测凝血酶

在玻碳电极（GCE）表面首先用增敏作用的多壁碳纳米管(MWCNTs)夹心于两层电沉积的铁氰化镍（NiHCF）氧化还原电化学探针之间,然后以金纳米粒子为固定核酸适配体的载体,构建了检测凝血酶的非标记型核酸适配体生物传感器。 利用扫描电子显微镜(SEM)对MWCNTs和NiHCF的形貌进行了表征。 利用电化学阻抗谱对传感器的组装过程进行了监测,用循环伏安法（CV）和差分脉冲伏安法（DPV）对传感器的电化学行为进行了研究。 以铁氰化镍为探针的传感器对凝血酶的检测在1.0 ng/L~1.0 mg/L范围内呈良好的线性关系,相关系数为0.998,检测限为0.2 ng/L(S/N=3)。     

Surface patterning using scanning electrochemical microscopy to locally trigger a “click” chemistry reaction

We report on the surface micropatterning of conductive surfaces via the electrochemical triggering of a click reaction, the copper(I) catalyzed azide–alkyne cycloaddition reaction (CuAAC) by SECM via a two-step approach: (i) functionalization on the entire surface with azido-aryl groups by using the diazonium approach followed by (ii) the covalent linkage of alkyne-bearing ferrocene by CuAAC within a local area by SECM. More precisely, the click reaction was triggered by Cu(I) catalyst generation for 30 min at the SECM tip positioned ≈ 10 μm above the azido-aryl modified surface. The dimension of the spot obtained under these conditions was ≈ 75 μm. The electrochemical imaging by SECM of the ultra thin area locally clicked with ferrocene moieties was made thanks to the electrocatalytic properties of the ferrocene modified surface towards ferrocyanide electrooxidation. This local clicking procedure opens the gate to further controlled functionalization of restricted small substrates.     

A comparison of four protocols for the immobilization of an aptamer on graphite composite electrodes

We have performed a comparative study on four protocols for the immobilization of the thrombin aptamer on a graphite-epoxy composite electrode with the aim to identify the most practical method for designing the corresponding impedimetric aptasensor. The protocols included (a) physical adsorption, (b) avidin-biotin affinity interaction, (c) electrochemical activation and covalent bonding via amide groups, and (d) electrochemical grafting using 4-carboxybenzenediazonium coupling. The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the (ferri/ferro)hexacyanide redox couple. An increase in the interfacial charge transfer resistance (R_ct) was noted in all cases after the aptamer-thrombin interaction had occurred. The selectivity of the aptasensor over common serum proteins was also systematically investigated. Physical adsorption resulted in the lowest detection limit of the probe (4.5 pM), while avidin-biotin interaction resulted in highest selectivity and reproducibility exhibiting a 4.9 % relative standard deviation at pM thrombin concentration levels.

Zeptomolar detection of Hg2 + based on label-free electrochemical aptasensor: One step closer to the dream of single atom detection

An ultra-sensitive and highly selective electrochemical label-free aptasensor is proposed for the quantitation of Hg^2 + based on the hybridization/dehybridization of double-stranded DNA (dsDNA) on a gold electrode. Thiol-substituted single-stranded DNA (ssDNA) is self-assembled on the gold electrode surface through the SAu interaction. The hybridization of ssDNA with complementary DNA (cDNA) and the consequences of dehybridization in the presence of mercury ions are followed through differential pulse voltammetry (DPV) responses using a [Fe(CN)₆]^{3 −/4 −} redox probe. The formation of a thymine–Hg^2 +–thymine (T–Hg^2 +–T) complex is the key to producing a highly selective and sensitive aptasensor for Hg^2 + determination. Specifically, the present electrochemical aptasensor is able to quantify Hg^2 + ions in concentrations from 5 zeptomolar (zM) to 55 picomolar (pM) with a limit of detection of 0.6 zM, close to the dream of single atom detection, without requiring a complicated procedure or expensive materials.     

A Label‐Free Electrochemical Aptasensor for Thrombin Using a Single‐Wall Carbon Nanotube (SWCNT) Casted Glassy Carbon Electrode (GCE)

An electrochemical aptasensor with a thrombin binding aptamer (TBA) was developed using a single‐wall carbon nanotube (SWCNT) casted GCE. The TBA was immobilized on SWCNTs through π‐stacking without any special modification, resulting in helical wrapping to the surface. In the presence of thrombin, the TBA binds with thrombin and the TBA concentration on the SWCNT surface decreases. The remaining amount of TBA can be analyzed by an electrochemical method without any label, because the guanine bases of the nucleic acid are measurable by electrochemical methods. The electrochemical oxidation of guanine nucleotides was enhanced by electrocatalytic mediation using Ru(bpy)₃²⁺ for higher sensitivity and reduction of the overpotential for electrochemical detection.     

Impedimetric Aptasensors Based on Carbon Nanotubes – Poly(methylene blue) Composite

The effect of aptamer structure and immobilization platform on the efficiency of thrombin binding and its detection using electrochemical impedance spectroscopy (EIS) characteristics was investigated with aptasensors based on glassy carbon electrodes covered with multiwalled carbon nanotubes (MWNTs). Aptamers with one or two binding sequences GGTTGGTGTGGTTGG specific for thrombin and poly(dA) and poly(dT) tags able to form dimeric products (aptabodies) were used to establish significance of steric and electrostatic factors in aptasensor performance. We have shown that electropolymerization of methylene blue onto MWNTs significantly improved electrochemical characteristics and sensitivity of thrombin detection against bare MWNTs. Charge transfer resistance and capacitance of the surface layer were measured in the presence of redox probe [Fe(CN)₆]^3?/4?. Aptasensors make it possible to detect thrombin in the concentration range 1 nM–1 µM with the limit of detection of 0.7 nM (monitoring resistance changes) and 0.5 nM (capacitance changes), respectively.     

Influence of surface oxygen groups on V(II) oxidation reaction kinetics

The role of surface oxygen groups on the kinetics of the V(II) oxidation reaction was studied on modified glassy carbon (GC) electrodes by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The reaction was found to be sensitive to the presence of oxygen groups on the electrode surface. Higher O/C ratios determined by X-ray photoelectron spectroscopy (XPS) corresponded to higher reactivities and lower charge transfer resistances measured in a 1 M V(II) electrolyte. The stability of an oxidised GC surface was also investigated in a 1 M V(II) electrolyte by potential holding and cycling experiments. It was found that after holding and cycling to successively more negative potentials up to − 0.8 V/RHE, the electrode surface lost its initial reactivity.     

Label-free aptasensor for thrombin using a glassy carbon electrode modified with a graphene-porphyrin composite

We report on an electrochemical aptasensor for the ultrasensitive determination of thrombin. A glassy carbon electrode modified with a graphene-porphyrin nanocomposite exhibits excellent electrochemical activity and can be used as a redox probe in differential pulse voltammetry of the porphyrin on its surface. The thrombin aptamer is then immobilized via p-stacking interactions between aptamer and graphene and π-π stacking with porphyrin simultaneously. The resulting electrochemical aptasensor displays a linear response to thrombin in the 5–1,500 nM concentration range and with a limit of detection of 0.2 nM (at an S/N of 3). The sensor benefits from the synergetic effects of graphene (with its high conductivity and high surface area), of the porphyrin (possessing excellent electrochemical activity), and of the aptamer (with its high affinity and specificity). This kind of aptasensor conceivably represents a promising tool for bioanalytical applications.

Mucin 4 detection with a label-free electrochemical immunosensor

Mucin 4 (MUC4) is a useful biomarker for endometriosis and cancers of the pancreas, esophagus and breast. The very first electrochemical immunosensor for the detection of MUC4 is reported, using carbon-based screen-printed electrodes modified by reaction with the diazonium salt of p-aminophenylacetic acid. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize and optimize the electrografting process. The in situ surface modification through diazotation with phenylacetic groups enables the chemical binding of the specific antibody, followed by its affinity reaction with MUC4. The immunosensor was optimized with respect to several parameters and is very promising for clinical applications, having a limit of detection of 0.33 μg mL^− 1 and a linear domain between 1 and 15 μg mL^− 1 obtained by electrochemical impedance spectroscopy measurements.     

Development of Electrochemical Impedance Spectroscopy Based Malaria Aptasensor Using HRP‐II as Target Biomarker

Current demand for a stable, low cost and sensitive malaria sensor has prompted to explore novel recognition systems that can substitute widely used protein based labile biorecognition elements to be used in point of care diagnostic devices. Here, we report a novel ssDNA aptamer of 90 mer sequence developed by SELEX process against HRP‐II, a specific biomarker for Plasmodium falciparum strains. High stability of the secondary structure of the isolated aptamer was discerned from its free energy of folding of −20.40 kcal mole⁻¹. The binding constant (K_d) of the aptamer with HRP‐II analysed by isothermal titration calorimetry was ∼1.32 μM. Circular dichroism studies indicated B form of the aptamer DNA. The aptamer was chemically immobilized on a gold electrode surface through a self‐assembled monolayer of dithio‐bis(succinimidyl) propionate to produce the aptasensor. The step wise modification of the layers over the gold electrode during fabrication of the aptasensor was confirmed by cyclic voltammetry. The aptasensor was then challenged with different concentration of HRP‐II and analysed the interaction signals through electrochemical impedance spectroscopy. The impedance signal behaved reciprocally with the increasing concentrations of the target in the sample from which a dynamic range of 1 pM–500 pM (R²=0.99) and LOD of ∼3.15 pM were discerned. The applicability of the developed aptasensor to detect HRP‐II in mimicked real sample was also validated.     

Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor

A bifunctional derivative of the thrombin-binding aptamer with a redox-active Fc moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent molecular beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin association could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidation signal to the increase in the thrombin concentration in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The molecular beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochemical signal upon subsequent thrombin binding.     

Label‐Free Electrochemical Aptasensor for Determination of Chloramphenicol Based on Gold Nanocubes‐Modified Screen‐Printed Gold Electrode

An ultrasensitive label‐free electrochemical aptasensor was developed for selective detection of chloramphenicol (CAP). The aptasensor was made using screen‐printed gold electrode modified with synthesized gold nanocube/cysteine. The interactions of CAP with aptamer were studied by cyclic voltammetry, square wave voltammetry (SWV) and electrochemical impedance spectroscopy. Under optimized conditions, two linear calibration curves were obtained for CAP determination using SWV technique, from 0.03 to 0.10 µM and 0.25–6.0 µM with a detection limit of 4.0 nM. The aptasensor has the advantages of good selectivity and stability and applied to the determination of CAP in human blood serum sample.     

G‐Quadruplex‐Based DNAzymes Aptasensor for the Amplified Electrochemical Detection of Thrombin

A novel G‐quadruplex‐based DNAzymes aptasensor for the amplified electrochemical detection of thrombin has been described. The aptasensor utilized a combination of hemin and guanine‐rich thrombin‐binding aptamer (TBA) to form horseradish peroxidase (HRP)‐mimicking DNAzymes with peroxidase catalytic activity. In the presence of thrombin, the enzyme activity could be extensively promoted, thereby providing the amplified electrochemical readout signals for detecting thrombin. This aptasensor exhibited high sensitivity and selectivity for thrombin determination, which enabled the analysis of thrombin with a detection limit of 6×10^–11 M. On the basis of results, this method could have broad applications in the detection of proteins and other biomolecules.     

Label-free and reagentless aptamer-based sensors for small molecules

A label free, reagentless aptasensor for adenosine is developed on an ISFET device. The separation of an aptamer/nucleic acid duplex by adenosine leads to the aptamer/adenosine complex that alters the gate potential of the ISFET. The sensitivity limit of the device is 5 x 10-5 M. Also, the immobilization of the aptamer/nucleic acid duplex on an Au-electrode and the separation of the duplex by adenosine mono-phosphate (AMP) enable the electrochemical detection of adenosine by faradaic impedance spectroscopy. The separation of the aptamer/nucleic acid duplex by adenosine and the formation of the aptamer/adenosine complex results in a decrease in the interfacial electron-transfer resistance in the presence of [Fe(CN)6]3-/4- as redox active substrate.