Direct electrochemical detection of individual collisions between magnetic microbead/silver nanoparticle conjugates and a magnetized ultramicroelectrode

Here, we report on the electrochemical detection of individual collisions between a conjugate consisting of silver nanoparticles (AgNPs) linked to conductive magnetic microbeads (cMμBs) via DNA hybridization and a magnetized electrode. The important result is that the presence of the magnetic field increases the flux of the conjugate to the electrode surface, and this in turn increases the collision frequency and improves the limit of detection (20 aM). In addition, the magnitude of the charge associated with the collisions is greatly enhanced in the presence of the magnetic field. The integration of DNA into the detection protocol potentially provides a means for using electrochemical collisions for applications in biological and chemical sensing.     

Target-driven self-assembly of stacking deoxyribonucleic acids for highly sensitive assay of proteins

In this paper, we report a new signal amplification strategy for highly sensitive and enzyme-free method to assay proteins based on the target-driven self-assembly of stacking deoxyribonucleic acids (DNA) on an electrode surface. In the sensing procedure, binding of target protein with the aptamer probe is used as a starting point for a scheduled cycle of DNA hairpin assembly, which consists of hybridization, displacement and target regeneration. Following numbers of the assembly repeats, a great deal of DNA duplexes can accordingly be formed on the electrode surface, and then switch on a succeeding propagation of self-assembled DNA concatemers that provide further signal enhancement. In this way, each target binding event can bring out two cascaded DNA self-assembly processes, namely, stacking DNA self-assembly, and therefore can be converted into remarkably intensified electrochemical signals by associating with silver nanoparticle-based readout. Consequently, highly sensitive detection of target proteins can be achieved. Using interferon-gamma as a model, the assay method displays a linear range from 1 to 500 pM with a detection limit of 0.57 pM, which is comparable or even superior to other reported amplified assays. Moreover, the proposed method eliminates the involvement of any enzymes, thereby enhancing the feasibility in clinical diagnosis.     

Electrochemical sensing of tumor suppressor protein p53–deoxyribonucleic acid complex stability at an electrified interface

Electrochemical biosensors have the unique ability to convert biological events directly into electrical signals suitable for parallel analysis. Here we utilize specific properties of constant current chronopotentiometric stripping (CPS) in the analysis of protein and DNA–protein complex nanolayers. Rapid potential changes at high negative current intensities (I_str) in CPS are utilized in the analysis of DNA–protein interactions at thiol-modified mercury electrodes. P53 core domain (p53CD) sequence-specific binding to DNA results in a striking decrease in the electrocatalytic signal of free p53. This decrease is related to changes in the accessibility of the electroactive amino acid residues in the p53CD–DNA complex. By adjusting I_str and temperature, weaker non-specific binding can be eliminated or distinguished from the sequence-specific binding. The method also reflects differences in the stabilities of different sequence-specific complexes, including those containing spacers between half-sites of the DNA consensus sequence. The high resolving power of this method is based on the disintegration of the p53CD–DNA complex by the electric field effects at a negatively charged surface and fine adjustment of the millisecond time intervals for which the complex is exposed to these effects. Picomole amounts of p53 proteins and DNA were used for the analysis at full electrode coverage but we show that even 10–20-fold smaller amounts can be analyzed. Our method cannot however take advantage of very low detection limits of the protein CPS detection because low I^_str intensities are deleterious to the p53CD–DNA complex stability at the electrode surface. These data highlight the utility of developing biosensors offering novel approaches for studying real-time macromolecular protein dynamics.     

Detecting DNA Damage with a Silver Solid Amalgam Electrode

Mercury electrodes modified with supercoiled (sc) DNA have been used as highly sensitive tools for the detection of DNA strand breaks or as sensors for DNA cleaving substances. In this paper we show that silver solid amalgam electrode (AgSAE), in connection with alternating current voltammetry, provides similar information about DNA damage as the hanging mercury drop electrode. The AgSAE can be used for the detection of enzymatic or chemical DNA cleavage in solution or at the electrode surface. AgSAE modified with scDNA can be utilized as a sensor for DNA nicking substances.     

Double‐Layer Nanogold and Double‐Strand DNA‐Modified Electrode for Electrochemical Immunoassay of Cancer Antigen 15‐3

Various sensor‐based immunoassay methods have been extensively developed for the detection of cancer antigen 15‐3 (CA 15‐3), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive electrochemical immunoassay for CA 15‐3 in human serum by using nanogold and DNA‐modified immunosensors. Prussian blue (PB), as a good mediator, was initially electrodeposited on a gold electrode surface, then double‐layer nanogold particles and double‐strand DNA (dsDNA) with the sandwich‐type architecture were constructed on the PB‐modified surface in turn, and then anti‐CA 15‐3 antibodies were adsorbed onto the surface of nanogold particles. The double‐layer nanogold particles provided a good microenvironment for the immobilization of biomolecules. The presence of dsDNA enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 1.0 to 240 ng/mL with a relatively low detection limit of 0.6 ng/mL (S/N=3) towards CA 15‐3. The stability, reproducibility and precision of the as‐prepared immunosensor were acceptable. 57 serum specimens were assayed by the developed immunosensor and standard enzyme‐linked immunosorbent assay (ELISA), respectively, and the results obtained were almost consistent. More importantly, the proposed methodology could be further developed for the immobilization of other proteins and biocompounds.     

Characterization of single-stranded DNA on chitosan-modified electrode and its application to the sequence-specific DNA detection

Single-strand DNA could bind with chitosan on a platinum electrode via forming a tight DNA-chitosan complex. The salt concentration of the ssDNA solution had an obvious effect on the surface coverage, the immobilization was remarkably reduced at high salt concentration. The sample ssDNA immobilized on the chitosan-modified electrode can hybridize efficiently with the complementary sequences and be successfully used for the sequence-specific DNA detection. The same results could be obtained using a gold or graphite electrode modified with chitosan. The stability of this electrode has been also discussed.     

Characterization of single-stranded DNA on chitosan-modified electrode and its application to the sequence-specific DNA detection

Single-strand DNA could bind with chitosan on a platinum electrode via forming a tight DNA-chitosan complex. The salt concentration of the ssDNA solution had an obvious effect on the surface coverage, the immobilization was remarkably reduced at high salt concentration. The sample ssDNA immobilized on the chitosan-modified electrode can hybridize efficiently with the complementary sequences and be successfully used for the sequence-specific DNA detection. The same results could be obtained using a gold or graphite electrode modified with chitosan. The stability of this electrode has been also discussed.     

Aptamer-based homogeneous protein detection using cucurbit[7]uril functionalized electrode

A new strategy for homogeneous protein detection is developed based on a cucurbit[7]uril (CB[7]) functionalized electrode. The analytical procedure consists of the binding of target protein to its aptamer in the test solution, followed by an exonuclease-catalyzed digestion of methylene blue (MB) tag labeled DNA oligonucleotides. Since CB[7] molecules immobilized on the electrode may efficiently capture the released MB-labeled nucleotides, the MB tags are concentrated to the electrode surface and subsequently yield highly sensitive electrochemical signal, which is related to the concentration of the target protein. The method combines the host–guest properties of CB[7] with the immobilization-free homogeneous assay, providing a powerful tool for protein detection. Taking the detection of osteopontin as an example, the proposed method can have a linear response to the target protein in a range from 50 to 500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹. It can also show high specificity and good reproducibility, and can be used directly for the assay of osteopontin in serum samples.     

Determination of Tetracycline at a UV‐Irradiated DNA Film Modified Glassy Carbon Electrode

In this study, interaction of tetracycline (TC) and DNA in the Britton? Robinson buffer solution (BR) was studied by cyclic voltammetry. The experimental results reveal that TC can bind strongly to DNA and the association constant and binding number between TC and DNA was obtained. Then DNA was immobilized on a glassy carbon electrode by UV‐irradiation. Through this process, water‐soluble DNA was converted into insoluble materials, and a stable DNA film was formed on the electrode. The electrochemical oxidation behavior of TC was studied at UV‐irradiated DNA film modified glassy carbon electrode (UV‐DNA‐GCE). The response of modified electrode was optimized with respect to pH, accumulation time, ionic strength, drug concentration and other variables. TC at the surface of modified electrode showed a linear dynamic range of 0.30–90.00 µM and a detection limit of 0.27 µM. To demonstrate the applicability of the modified electrode, it was used for the analysis of real samples such as pharmaceutical formulations and milk.     

Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

We present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.     

An Impedimetric Aptasensor Based on a Novel Line‐Pad‐Line Electrode for the Determination of VEGF165

In this work, a novel, simple and label‐free line‐pad‐line electrode (LPLE) biosensor was developed for detection of vascular endothelial growth factor (VEGF₁₆₅). DNA aptamer was used as a recognition element for high specificity to VEGF₁₆₅, and original LPLE as the substrate electrode for high sensitivity of the biosensor. This sensor was prepared by immobilizing anti‐VEGF₁₆₅ aptamers on the LPLE surface through gold‐sulfur (Au?S) bonding. Upon the addition of VEGF₁₆₅, a large target‐induced conformational change in the surface‐immobilized aptamer was generated and caused variations in the interfacial properties,which led to a corresponding increase in the impedance magnitude of the LPLE. Finally, our results demonstrate that the calibration curve for VEGF₁₆₅determination was linear over the range of 0.026‐31.4 fM with a detection limit as low as 0.017 fM.Additionally, our sensor was fabricated on printed circuit board (PCB) with a new electrode construction, and can potentially be implemented with the advantages of simplicity, low‐cost and easy mass production. Besides, considering its desirable sensitivity and specificity, the proposed use of LPLE provided a promising strategy for a wide variety of sensing applications.     

Target induced dissociation (TID) strategy for the development of electrochemical aptamer-based biosensor

In this paper, we report a novel and more general signal-on strategy for the fabrication of electrochemical aptamer-based (E-AB) biosensor. The principle is that the interaction between the target and the aptamer strand may induce the formation and subsequent dissociation of target–aptamer complex from an electrode surface, and consequently, the remaining DNA strand on the electrode surface can hybridize again with a ssDNA containing an electrochemical probe. Differential pulse voltammetric studies have revealed that this target induced disassociation (TID) strategy is an effective signal-on method for the detection of ATP molecules with good selectivity. The TID strategy may also have several advantages, such as independence on the specific structure of either the aptamers or their complementary sequences and promotion of the generalization of E-AB sensors, the more convincible results due to the signal-on model, and the unnecessity to label the aptamers, which provides the optimized status for the reaction with the targets, etc.     

High-throughput imaging assay of multiple proteins via target-induced DNA assembly and cleavage

This work integrates target-induced DNA assembly and cleavage on a DNA chip to design a versatile imaging strategy as an assay for multiple proteins. The DNA assembly is achieved via immunological recognition to trigger the proximity hybridization for releasing a DNA sequence, which then hybridizes with FITC-DNA1 immobilized on the chip to induce the enzymatic cleavage of DNA1 and thus decrease the signals. The signal readout is performed with both fluorescent imaging of the left FITC and chemiluminescent (CL) imaging, by adding peroxidase labelled anti-FITC in assembly solution and CL substrates to produce CL emission. This one-step incubation can be completed in 30 min. The imaging method shows wide detection ranges and detection limits down to pg mL^–1 for the simultaneous detection of 4 protein biomarkers. This high-throughput strategy with good practicability can be easily extended to other protein analytes, providing a powerful protocol for protein analysis and clinical diagnosis.     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.     

检测痕量Hg~(2+)的DNA电化学生物传感器

通过自组装方法将修饰有二茂铁基团的富T序列DNA分子(DNA-Fc)固定在金电极表面,得到了一种基于DNA修饰电极的电化学汞离子(Hg2+)传感器.当溶液中有Hg2+存在时,Hg2+可与修饰电极上DNA的T碱基发生较强的特异结合,形成T-Hg2+-T发卡结构,使DNA分子构象发生改变,其末端具有电化学活性的二茂铁基团远离电极表面,电化学响应随之发生变化.示差脉冲伏安法(DPV)结果显示:DNA末端二茂铁基团的还原峰在0.26V(vs饱和甘汞电极(SCE))附近,峰电流随溶液中Hg2+浓度的增加而降低;Hg2+浓度范围在0.1nmol·L-1-1μmol·L-1时,电流相对变化率与Hg2+浓度的对数呈现良好的线性关系.该修饰电极对Hg2+的检测限为0.1nmol·L-1,可作为痕量Hg2+检测的电化学生物传感器.干扰实验也表明,该传感器对Hg2+具有良好的特异性与灵敏度.     

Detection of DNA immobilized on bare gold electrodes and gold nanoparticle-modified electrodes via electrogenerated chemiluminescence using a ruthenium complex as a tag

Electrogenerated chemiluminescence (ECL) for DNA hybridization detection is demonstrated based on DNA that was self-assembled onto a bare gold electrode and onto a gold nanoparticles modified gold electrode. A ruthenium complex served as an ECL tag. Gold nanoparticles were self-assembled on a gold electrode associated with a 1,6-hexanedithiol monolayer. The surface density of single stranded DNA (ssDNA) on the gold nanoparticle modified gold electrode was 4.8?×?10¹⁴ molecules per square centimeter which was 12-fold higher than that on the bare gold electrode. Hybridization was induced by exposure of the target ssDNA gold electrode to the solution of ECL probe consisting of complementary ssDNA tagged with ruthenium complex. The detection limit of target ssDNA on a gold nanoparticle modified gold electrode (6.7?×?10^?12 mol L^?1) is much lower than that on a bare gold electrode (1.2?×?10^?10 mol L^?1). The method has been applied to the detection of the DNA sequence related to cystic fibrosis. This work demonstrates that employment of gold nanoparticles self-assembled on a gold electrode is a promising strategy for the enhancement of the sensitivity of ECL detection of DNA.     

Detection of DNA and Protein Molecules Using an FET‐Type Biosensor with Gold as a Gate Metal

In this study, a field effect transistor (FET)‐type biosensor based on 0.5 μm standard complementary metal oxide semiconductor (CMOS) technology is proposed and its feasibility for detecting deoxyribonucleic acid (DNA) and protein molecules is investigated. Au, which has a chemical affinity with thiol by forming a self‐assembled monolayer (SAM), was used as the gate metal in order to immobilize DNA and protein molecules. A Pt pseudo‐reference electrode was employed for the detection of biomolecules. The sensor was fabricated as a p‐channel (P)MOSFET‐type because PMOSFET with positive surface potential is useful for detecting negatively charged biomolecules from the view point of its high sensitivity and fast response time. DNA and protein molecules were detected by measuring the variation of the drain current due to the variation of biomolecular charge and capacitance. DNA and protein molecules used in the experiment were 15mer–oligonucleotide probe and streptavidin‐biotin protein complexes, respectively. DNA was detected by both in situ and ex situ measurements. Additionally, to verify the interactions among SAM, streptavidin, and biotin, surface plasmon resonance (SPR) measurement was performed.     

Electrochemical Behavior of 8‐Azaguanine at DNA Langmuir–Blodgett Modified Glassy Carbon Electrode and Its Analytical Application

A highly sensitive electrochemical biosensor for the detection of trace amounts of 8‐azaguanine has been designed. Double stranded (ds)DNA molecules are immobilized onto a glassy carbon electrode surface with Langmuir–Blodgett technique. The adsorptive voltammetric behaviors of 8‐azaguanine at DNA‐modified electrode were explored by means of cyclic voltammetry and square wave voltammetry. Compared with bare glassy carbon electrode (GCE), the Langmuir–Blodgett film modified electrode can greatly improve the measuring sensitivity of 8‐azaguanine. Under the optimum experimental conditions, the Langmuir–Blodgett film modified electrode in pH 3.0 Britton–Robinson buffer solutions shows a linear voltammetric response in the range of 5.0×10^?8 to 1.0×10^?5 mol L^?1 with detection limit 9.0×10^?9 mol L^?1. The method proposed was applied successfully for the determination of 8‐azaguanine in diluted human urine with wonderful satisfactory.     

Determination of radon using solid state nuclear tracks wireless sensing method

A highly sensitive and selective electrochemiluminescent (ECL) biosensor for the determination of adenosine was developed. Single DNA (capture DNA) was immobilized on the gold electrode through Au-thiol interaction at first. Another DNA modified with tris(2,2'-bipyridyl) ruthenium(II)-doped silica nanoparticles (Ru-SNPs) that contained adenosine aptamer was then modified on the electrode surface through hybridizing with the capture DNA. In the presence of adenosine, adenosine-aptamer complex is produced rather than aptamer-DNA duplex, resulting with the dissociation of Ru-SNPs-labeled aptamer from the electrode surface and the decrease in the ECL intensity. The decrease of ECL intensity has a direct relationship with the logarithm of adenosine concentration in the range of 1.0×10(-10) to 5.0×10(-6)molL(-1). The detection limit of the proposed method is 3.0×10(-11)molL(-1). The existence of guanosine, cytidine and uridine has little interference with adenosine detection, demonstrating that the developed biosensor owns a high selectivity to adenosine. In addition, the developed biosensor also demonstrates very good reusability, as after being reused for 30 times, its ECL signal still keeps 91% of its original state.     

Electrochemical DNA biosensor for human papillomavirus 16 detection in real samples

An electrochemical DNA biosensor for human papillomavirus (HPV) 16 detection has been developed. For this proposed biosensor, l-cysteine was first electrodeposited on the gold electrode surface to form l-cysteine film (CYSFILM). Subsequently, HPV16-specific probe was immobilized on the electrode surface with CYSFILM. Electrochemistry measurement was studied by differential pulse voltammetry method (DPV). The measurement was based on the reduction signals of methylene blue (MB) before and after hybridization either between probe and synthetic target or extracted DNA from clinical samples. The effect of probe concentration was analyzed and the best results were seen at 1000 nM. The hybridization detection presented high sensitivity and broad linear response to the synthetic-target concentration comprised between 18.75 nM and 250 nM as well as to a detection limit of 18.13 nM. The performance of this biosensor was also investigated by checking probe-modified electrode hybridization with extracted DNA from samples. The results showed that the biosensor was successfully developed and exhibited high sensitivity and satisfactory selectivity to HPV16. These results allow for the possibility of developing a new portable detection system for HPVs and for providing help in making an effective diagnosis in the early stages of infection.