Amplified detection of single-base mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction

Three different methods for the amplified detection of a single-base mismatch in DNA are described using microgravimetric quartz-crystal-microbalance as transduction means. All methods involve the primary incorporation of a biotinylated base complementary to the mutation site in the analyzed double-stranded primer/DNA assembly. The double-stranded assembly is formed between 25 complementary bases of the probe DNA assembled on the Au-quartz crystal and the target DNA. One method of amplification includes the association of avidin- and biotin-labeled liposomes to the sensing interface. The second method of amplified detection of the base mismatch includes the association of an Au-nanoparticle-avidin conjugate to the sensing interface, and the secondary Au-nanoparticle-catalyzed deposition of gold on the particles. The third amplification route includes the binding of the avidin-alkaline phosphatase biocatalytic conjugate to the double-stranded surface followed by the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate to the insoluble product indigo derivative that precipitates on the transducer. Comparison of the three amplification routes reveals that the catalytic deposition of gold on the Au-nanoparticle/avidin conjugate is the most sensitive method, and the single-base mismatch in the analyzed DNA is detected with a sensitivity that corresponds to 3x10(-16) M.     

Nanostructured Screen Printed Graphite Electrode for the Development of a Novel Electrochemical Genosensor

The aim of this work is the preparation of DNA‐sensing architectures based on gold nanoparticles (AuNPs) in conjunction with an enzyme‐amplified detection to improve the analytical properties of genosensor. In order to assess the utility of study as DNA‐sensing devices, a thiolated DNA capture probe sequence was immobilized on the gold nanoparticle modified surface. After labeling of the biotinylated hybrid with a streptavidin‐alkaline phosphatase conjugate, the electrochemical detection of the enzymatic product was performed on the surface of a disposable electrode. Two different enzymatic substrates to detect the hybridization event were studied. In the first case, the enzyme catalyzed the hydrolysis of α‐naphthyl phosphate; the product is electroactive and has been detected by means of differential pulse voltammetry (DPV). In the second one, the enzyme catalyzed the precipitation of an insoluble and insulating product on the sensing interface. In this case, the electrochemical transduction of the hybridization process was performed by electrochemical impedance spectroscopy (EIS).     

基于酶催化沉积质量放大的压电免疫传感器的研究

提出了一种酶催化沉积质量放大的高灵敏压电免疫传感器. 采用1,6-二巯基己烷在石英晶振上自组装一单分子层, 再通过另一端巯基连接胶体金, 利用胶体金的高比表面积和强吸附作用力增加抗IgG抗体的固定量, 同时借助胶体金优良的生物亲和性保持抗IgG抗体的活性. 在H2O2存在下, 通过标记在抗人IgG抗体上的HRP酶催化底物DAB(3,3′-联苯二胺), 反应中生成的不溶性产物沉积到石英晶振的Au电极表面, 达到质量放大的目的. 结果表明, 检测人IgG在16 ng/mL-100 μg/mL范围内有很好的线性关系, 检测下限为10 ng/mL, 在用于实际试样的回收率测定中, 结果良好.     

Probing Biomolecular Interactions at Conductive and Semiconductive Surfaces by Impedance Spectroscopy: Routes to Impedimetric Immunosensors,DNA‐Sensors,and Enzyme Biosensors

Impedance spectroscopy is a rapidly developing electrochemical technique for the characterization of biomaterial‐functionalized electrodes and biocatalytic transformations at electrode surfaces, and specifically for the transduction of biosensing events at electrodes or field‐effect transistor devices. The immobilization of biomaterials, e.g., enzymes, antigens/antibodies or DNA on electrodes or semiconductor surfaces alters the capacitance and interfacial electron transfer resistance of the conductive or semiconductive electrodes. Impedance spectroscopy allows analysis of interfacial changes originating from biorecognition events at electrode surfaces. Kinetics and mechanisms of electron transfer processes corresponding to biocatalytic reactions occurring at modified electrodes can be also derived from Faradaic impedance spectroscopy. Different immunosensors that use impedance measurements for the transduction of antigen‐antibody complex formation on electronic transducers were developed. Similarly, DNA biosensors using impedance measurements as readout signals were developed. Amplified detection of the analyte DNA using Faradaic impedance spectroscopy was accomplished by the coupling of functionalized liposomes or by the association of biocatalytic conjugates to the sensing interface providing biocatalyzed precipitation of an insoluble product on the electrodes. The amplified detections of viral DNA and single‐base mismatches in DNA were accomplished by similar methods. The changes of interfacial features of gate surfaces of field‐effect transistors (FET) upon the formation of antigen‐antibody complexes or assembly of protein arrays were probed by impedance measurements and specifically by transconductance measurements. Impedance spectroscopy was also applied to characterize enzyme‐based biosensors. The reconstitution of apo‐enzymes on cofactor‐functionalized electrodes and the formation of cofactor‐enzyme affinity complexes on electrodes were probed by Faradaic impedance spectroscopy. Also biocatalyzed reactions occurring on electrode surfaces were analyzed by impedance spectroscopy. The theoretical background of the different methods and their practical applications in analytical procedures were outlined in this article.     

Enzyme-catalyzed amplified immunoassay for the detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>-specific IgG using Faradaic impedance spectroscopy,CV and QCM

A highly sensitive electrochemical immunoassay for Toxoplasma gondii-specific IgG (Tg-IgG) in human serum has been developed that is based on an enzyme-catalyzed amplification due to the formation of an insoluble precipitate on the surface of a quartz crystal microbalance (QCM). T. gondii antigen (TgAg) was immobilized on the surface of a gold electrode in order to bind Tg-IgG, and this was followed by the addition of anti-Tg-IgG horseradish peroxidase conjugate (anti-Tg-IgG-HRP). Subsequent exposure to 3,3-diaminobenzidine (DAB) led to the enzymatically-catalyzed amplified deposition of the oxidation products on the QCM surface in the presence of H₂O₂. The transduction methods electrochemical Faradaic impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to assay the resistance to electron transfer at the conductive support upon accumulation of the insoluble products. The precipitation process was monitored in real time by QCM. The assay conditions, including the concentration of immobilized TgAg and the dosage of anti-Tg-IgG-HRP conjugate, were optimized. It was found that the amount of precipitate that accumulated on the conductive QCM surface was determined by the concentration of the target analyte Tg-IgG and the time permitted for biocatalyzed precipitation. The technique was shown to give a linear electron transfer resistance response (as measured by EIS) for Tg-IgG dilutions ranging between 1:8000 and 1:200, and a detection limit of 1:9600 dilution.     

Amplified Electrochemical DNA Sensor Based on Polyaniline Film and Gold Nanoparticles

In this work, an electrochemical DNA biosensor, based on a dual signal amplified strategy by employing a polyaniline film and gold nanoparticles as a sensor platform and enzyme‐linked as a label, for sensitive detection is presented. Firstly, polyaniline film and gold nanoparticles were progressively grown on graphite screen‐printed electrode surface via electropolymerization and electrochemical deposition, respectively. The sensor was characterized by scanning electron microscopy (SEM), cyclic voltammetry and impedance measurements. The polyaniline‐gold nanocomposite modified electrodes were firstly modified with a mixed monolayer of a 17‐mer thiol‐tethered DNA probe and a spacer thiol, 6‐mercapto‐1‐hexanol (MCH). An enzyme‐amplified detection scheme, based on the coupling of a streptavidin‐alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalyzed the hydrolysis of the electroinactive α‐naphthyl phosphate to α‐naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. In this way, the sensor coupled the unique electrical properties of polyaniline and gold nanoparticles (high surface area, fast heterogeneous electron transfer, chemical stability, and ease of miniaturisation) and enzymatic amplification. A linear response was obtained over a concentration range (0.2–10 nM). A detection limit of 0.1 nM was achieved.     

Detection of hydrogen peroxide and glucose by enzyme product precipitation on sensor surface

Biocatalysed precipitation of an insoluble product accumulated on the enzyme-modified electrode surface was applied as the amplification path for low concentration sensing of hydrogen peroxide and glucose. Sensitive electrochemical and quartz-crystal microbalance (QCM) biosensors based on biocatalytic precipitation were developed. A horseradish peroxidase (HRP) monolayer-modified electrode was used to sense H₂O₂ via the oxidation of 4-chloro-1-naphthol (4CN) forming insoluble benzo-4-chlorocyclohexadienone. Additionally, the bienzyme system employed glucose oxidase (GOx) linked to HRP/4CN. The amount of the precipitate assembled on the sensing surface corresponded to the concentration of analytes and to the length of the incubation interval. The precipitated deposits were followed as a change of impedance using cyclic voltammetry (CV), mass change was determined continuously using a microgravimetric quartz-crystal microbalance, and optical microscopy enabled the visualisation of the precipitate. Regeneration of the enzyme-modified electrode was performed using cathodic reduction of the insoluble product. Thus, a simple biosensor for multiple analyses with low detection limits and of low cost can be developed.     

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.     

Au nanoparticle-enhanced surface plasmon resonance sensing of biocatalytic transformations

N-(3-Aminopropyl)-N'-methyl-4,4'-bipyridinium is coupled to tiopronin-capped Au nanoparticles (diameter ca. 2 nm) to yield methyl(aminopropyl)viologen-functionalized Au nanoparticles (MPAV(2+)-Au nanoparticles). In situ electrochemical surface plasmon resonance (SPR) measurements are used to follow the electrochemical deposition of the bipyridinium radical cation modified Au nanoparticles on an Au-coated glass surface and the reoxidation and dissolution of the bipyridinium radical cation film. The MPAV(2+)-functionalized Au nanoparticles are also employed for the amplified SPR detection of NAD(+) and NADH cofactors. By SPR monitoring the partial biocatalyzed dissolution of the bipyridinium radical cation film in the presence of diaphorase (DP) NAD(+) is detected in the concentration range of 1x10(-4) M to 2x10(-3) M. Similarly, the diaphorase-mediated formation of the bipyridinium radical cation film on the Au-coated glass surface by the reduction of the MPAV(2+)-functionalized Au nanoparticles by NADH is used for the amplified SPR detection of NADH in the concentration range of 1x10(-4) M to 1x10(-3) M.     

DNA analysis by application of Pt nanoparticle electrochemical amplification with single label response

This study demonstrates a highly sensitive sensing scheme for the detection of low concentrations of DNA, in principle down to the single biomolecule level. The previously developed technique of electrochemical current amplification for detection of single nanoparticle (NP) collisions at an ultramicroelectrode (UME) has been employed to determine DNA. The Pt NP/Au UME/hydrazine oxidation reaction was employed, and individual NP collision events were monitored. The Pt NP was modified with a 20-base oligonucleotide with a C6 spacer thiol (detection probe), and the Au UME was modified with a 16-base oligonucleotide with a C6 spacer thiol (capture probe). The presence of a target oligonucleotide (31 base) that hybridized with both capture and detection probes brought a Pt NP on the electrode surface, where the resulting electrochemical oxidation of hydrazine resulted in a current response.     

Dendritic-like streptavidin/alkaline phosphatase nanoarchitectures for amplified electrochemical sensing of DNA sequences

This study describes the development and characterization of a novel dendritic-like signal amplification pathway. Such an analytical strategy relies on the use of streptavidin and biotinylated alkaline phosphatase, which can be simply and conveniently self-assembled to build nanoarchitectures rich in enzyme labels.The performance of this enzyme-based amplification route was demonstrated in connection with the electrochemical sensing of DNA sequences. Compared to the commercially available streptavidin-conjugated alkaline phosphatase labels, a single generation of the streptavidin/biotinylated alkaline phosphatase assembly allowed a 15-20-fold enhancement of the electroanalytical signals. The higher sensitivity allowed by the dendritic-like route was attributed to the lower steric hindrance of the proteins employed for this amplification path. As low as 50 pmol/L of a 388-bp-long amplicon identifying Salmonella spp. was easily detected. The experimental results additionally demonstrated that the sensitivity of the method could be further increased in a linear fashion with the number of protein-enzyme generations.     

An electrochemical immunosensor using p-aminophenol redox cycling by NADH on a self-assembled monolayer and ferrocene-modified Au electrodes

Redox cycling of enzymatically amplified electroactive species has been widely employed for high signal amplification in electrochemical biosensors. However, gold (Au) electrodes are not generally suitable for redox cycling using a reducing (or oxidizing) agent because of the high background current caused by the redox reaction of the agent at highly electrocatalytic Au electrodes. Here we report a new redox cycling scheme, using nicotinamide adenine dinucleotide (NADH), which can be applied to Au electrodes. Importantly, p-aminophenol (AP) redox cycling by NADH is achieved in the absence of diaphorase enzyme. The Au electrodes are modified with a mixed self-assembled monolayer of mercaptododecanoic acid and mercaptoundecanol, and a partially ferrocenyl-tethered dendrimer layer. The self-assembled monolayer of long thiol molecules significantly decreases the background current of the modified Au electrodes, and the ferrocene modification facilitates easy oxidation of AP. The low amount of ferrocene on the Au electrodes minimizes ferrocene-mediated oxidation of NADH. In sandwich-type electrochemical immunosensors for mouse immunoglobulin G (IgG), an alkaline phosphatase label converts p-aminophenylphosphate (APP) into electroactive AP. The amplified AP is oxidized to p-quinoneimine (QI) by electrochemically generated ferrocenium ion. NADH reduces QI back to AP, which can be re-oxidized. This redox cycling enables a low detection limit for mouse IgG (1 pg mL(-1)) to be obtained.     

A biotinylated intercalator for selective post-labeling of double-stranded DNA as a basis for high-sensitive DNA assays

For increasing the sensitivity of label-free DNA assays an amplification strategy is proposed based on the synthesis of a proflavine derivative which on the one hand retains its high affinity for double-stranded DNA (dsDNA) intercalation and on the other hand is functionalized via a flexible spacer with biotin moieties. By this, subsequent to the post-labeling of areas with dsDNA, reporter systems such as streptavidin/enzyme conjugates can be bound. Amplified DNA hybridization detection using an oligonucleotide model system, a biotinylated proflavine as intercalator and streptavidin/alkaline phosphatase is demonstrated.     

Probing kinase activities by electrochemistry, contact-angle measurements, and molecular-force interactions

Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region.     

Electronic transduction of DNA sensing processes on surfaces: amplification of DNA detection and analysis of single-base mismatches by tagged liposomes.

Tagged, negatively charged, liposomes are used to amplify DNA sensing processes. The analyses of the target DNA are transduced electrochemically by using Faradaic impedance spectroscopy, or by microgravimetric measurements with Au-quartz crystals. By one method, a probe oligonucleotide (1) is assembled on Au-electrodes or Au-quartz crystals. The formation of the double-stranded assembly with the analyte DNA (2) is amplified by the association of the 3-oligonucleotide-functionalized liposomes to the sensing interface. The target DNA is analyzed by this method with a sensitivity limit that corresponds to 1 x 10(-12) M. A second method to amplify the sensing of the analyte involves the interaction of the 1-functionalized electrode or Au-quartz crystal with the target DNA sample (2) that is pretreated with the biotinylated oligonucleotide (4). The formation of the three-component double-stranded assembly between 1/2/4 is amplified by the association of avidin and biotin-labeled liposomes to the sensing interfaces. By the secondary association of avidin and biotin-tagged liposomes, a dendritic-type amplification of the analysis of the DNA is accomplished. The analyte DNA (2) is sensed by this method with a sensitivity limit corresponding to 1 x 10(-13) M. The biotin-tagged liposomes are also used to probe and amplify single-base mismatches in an analyte DNA. The 6-oligonucleotide-functionalized Au-electrode or Au-quartz crystal was used to differentiate the single-base mismatch (G) in the mutant (5) from the normal A-containing gene (5a). Polymerase-induced coupling of the biotinylated-C-base to the double-stranded assembly generated between 6 and 5 followed by the association of avidin and biotin-tagged liposomes is used to probe the single base mismatch. The functionalized liposomes provide a particulate building unit for the dendritic amplification of DNA sensing.     

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase.In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection.Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.     

Genomagnetic electrochemical assays of DNA hybridization

An electrochemical genomagnetic hybridization assay has been developed to take advantage of a new and efficient magnetic separation/mixing process, the amplification feature of enzyme labels, and single-use thick-film carbon transducers operated in the pulse-voltammetric mode. It represents the first example of coupling a magnetic isolation with electrochemical detection of DNA hybridization. The new protocol employs an enzyme-linked sandwich solution hybridization, with a magnetic-particle labeled probe hybridizing to a biotinylated DNA target that captures a streptavidin-alkaline phosphatase (AP). The alpha-naphthol product of the enzymatic reaction is quantitated through its well-defined, low-potential (+0.1 V vs. Ag/AgCl) differential pulse-voltammetric peak at the disposable screen-printed electrode. The efficient magnetic isolation is particularly attractive for electrical detection of DNA hybridization which is commonly affected by the presence of non-hybridized nucleic acid adsorbates. The new biomagnetic processing combines such magnetic separation with a low-volume magnetic mixing, and allows simultaneous handling of 12 samples. The attractive bioanalytical behavior of the new enzyme-linked genomagnetic electrical assay is illustrated for the detection of DNA segments related to the breast-cancer BRCA1 gene.     

Magnetically trigged direct electrochemical detection of DNA hybridization using Au67 quantum dot as electrical tracer

A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.     

4-Amino-1-naphthylphosphate as a substrate for the amperometric detection of alkaline phosphatase activity and its application for immunoassay

Immunosensors and biochemical array detection systems based on electrochemical transducers have many advantages such as low detection limit, fast response, simple design and ease of miniaturization. However, further development of such sensors will depend on the availability of suitable substrates that can be converted by a labeling enzyme to an electrochemically active product. Here, we report the synthesis of 4-amino-1-naphthylphosphate and it’s application as a new substrate for alkaline phosphatase. The electrochemical and enzymatic properties of this compound were investigated and compared with the properties of other aromatic 1,4-dihydroxy and 1,4-hydroxy-amine derivatives. The product of the enzyme reaction was 4-aminonaphthol, which was rapidly converted in the presences of air to 1,4-iminonaphthoquinone. This compound could then be detected in an amperometric flow injection assay (AFIA) with −200 mV versus Ag/AgCl potential application. The analytical range for mouse IgG, in an alkaline phosphatase amplified sandwich immuoassay with amperometric detection, was 0.01-100 μg ml⁻¹.     

滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

通过捕获探针与纳米金膜之间的共价连接, 保证了滚环扩增(RCA)产物始终结合于金膜表面, Phi29 DNA聚合酶的高效扩增和Escherichia coli DNA链接酶的高度精确性使检测达到单碱基识别, 检测灵敏度达到10⁴ copies/mL. 实验结果表明, 与单碱基错配序列相比, RCA可明显增强检测的灵敏度. 该RCA基因传感器操作简单, 灵敏度和特异性较高, 在乙型肝炎病毒的快速检测方面具有一定的开发潜力.