Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Characteristics of DNA during binding with polycyclic aromatic hydrocarbons in vitro]

Polycyclic aromatic hydrocarbons (PAH) were covalently bound to DNA by means of various activating systems. The following systems were used: the microsomal fraction of the rat liver, the system with I2, the system with ascorbic acid and FeSO4. Breaks in DNA due to the activating systems action appeared in all of these systems. Plateau of the PAH binding system curve in the microsomal system cannot be attributed either to the fall of the PAH metabolism rate to zero, or to the PAH binding sites in DNA. This plateau is the result of equalization of the rates of the two contrary-directed processes: the binding of metabolites and their removal due to DNA degradation. Because of the breaks in DNA caused by the activating systems, the authors failed to discover the changes in sedimentation data of DNA due to the covalently bound PAH.     

Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode

Sequence-known short-stranded hepatitis B virus (HBV) DNA fragment (181 bps) was obtained by PCR method. The strategy for its electrochemical detection was designed by covalently immobilizing single-stranded HBV DNA on gold electrode surface via carboxylate ester as a linkage between 3′-hydroxy end of DNA and carboxyl group of thioglycolic acid (TGA) self-assembled monolayer. The hybridization reaction on surface was evidenced by electrochemical methods using ferrocenium hexafluorophosphate (FcPF₆) as an electroactive indicator. The interactions of Fc⁺ with single-stranded (ss) and double-stranded (ds) HBV DNA immobilized on TGA monolayer were studied. The difference between the responses of Fc⁺ at ss- and ds-DNA/Au electrodes suggested that this hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. AC impedance and XPS techniques have been employed to characterize the immobilization of ss-DNA on the gold surface.     

Hybridization biosensor using di(2,2'-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3(')-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy)(2)Cl(2)](+) as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy)(2)Cl(2)](+) in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy)(2)Cl(2)](+) at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.     

Electric potential control of DNA immobilization on gold electrode

The assembly of synthetic, controllable molecules is one of the goals in nanotechnology. The primary objective of this contribution is to selectively immobilize DNA on gold via electric potential control. The self-assembly monolayer (SAM) was prepared with 2-aminoethanethiol (AET) on the gold electrode. A new approach based on electric potential was firstly used to control DNA immobilization covalently onto the SAM with the activation of 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) in low ionic strength solution. The influence of electric potential on DNA immobilization was investigated by means of cyclic voltammogram, A.C. impedance, auger electron spectrometer as well as atomic force microscope (AFM) on template-stripped gold surface. The result proves that controlled potential can affect the course of DNA immobilization. More negative potential can restrain the DNA immobilization, while the more positive potential can accelerate the DNA immobilization. It is of great significance for the control of DNA self-assembly and will find wide application in the fields of DNA-based devices.     

DNA probes on chip surfaces studied by scanning force microscopy using specific binding of colloidal gold

Single-stranded DNA was covalently bound on chip surfaces using two different silanization procedures. The resulting surfaces were characterized by fluorescence and scanning force microscopy using sequence-complementary DNA molecules with labels. Colloidal gold (30 nm) was used as the topographic label. Scanning force microscopy revealed the individual labels on the surface and their distribution. Steps of silane layers or DNA-modified surfaces prepared using an elastomeric mask provided internal controls for comparison of modified with unmodified surfaces.     

Coupling of a DNA piezoelectric biosensor and polymerase chain reaction to detect apolipoprotein E polymorphisms

In this paper we report the coupling of the Polymerase Chain Reaction (PCR) with a piezoelectric biosensor to detect a point mutation in a human gene. Biotinylated 23-mer probes were immobilised on the streptavidin coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridisation of the immobilised probes with a short sequence (23 mer) complementary, non-complementary and mismatched DNA was investigated: the device was able to distinguish the different synthetic oligonucleotides. Many cycles of measurements can be performed on the same crystal surface regenerating the single strand of DNA with 1 mM of HCl. The same hybridisation reaction was then performed using real samples of human DNA extracted from blood and amplified by PCR, following a standard procedure for genetic detection of the polymorphism of the apolipoprotein E (apoE) gene. The procedure was able to distinguish the sequences present in the different samples, which differ only in one base: in this way it was possible distinguish between different groups of genotypes with apoE typing. Experiments with 'blank' samples confirmed the absence of adsorption or non-specific effects on the quartz crystal treated with the reported procedure.     

A doubly amplified electrochemical immunoassay for carcinoembryonic antigen

An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody-glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.     

Spin-on end-functional diblock copolymers for quantitative DNA immobilization

We demonstrate a simple means to covalently bond DNA to both hard (i.e., glass and silicon wafers) and soft (i.e., polymeric) substrates that provides quantitative and precise control of the DNA areal density. The approach is based on spin coating an alkyne-end-functional diblock copolymer, alpha-alkyne-omega-Br-poly( tBA- b-MMA), that self-assembles on both types of substrates as an ordered monolayer and thereby directs alkyne groups to the surface. Azido-functionalized DNA is covalently linked to the alkyne functionalized substrates by means of a "click" reaction between azide and alkyne groups. The density of immobilized DNA can be quantitatively controlled by varying the parameters used for spin-coating the copolymer film, that is, solution concentration and rotational speed, or by varying the copolymer molecular weight. We find the yield of the DNA coupling reaction to be dependent on the nature of the polymer underlying the reactive alkyne functional groups, being higher for more hydrophilic polymers.     

Development of a multilayered polymeric DNA biosensor using radio frequency technology with gold and magnetic nanoparticles

This study utilized the radio frequency (RF) technology to develop a multilayered polymeric DNA sensor with the help of gold and magnetic nanoparticles. The flexible polymeric materials, poly (p-xylylene) (Parylene) and polyethylene naphtholate (PEN), were used as substrates to replace the conventional rigid substrates such as glass and silicon wafers. The multilayered polymeric RF biosensor, including the two polymer layers and two copper transmission structure layers, was developed to reduce the total sensor size and further enhance the sensitivity of the biochip in the RF DNA detection. Thioglycolic acid (TGA) was used on the surface of the proposed biochip to form a thiolate-modified sensing surface for DNA hybridization. Gold nanoparticles (AuNPs) and magnetic nanoparticles (MNPs) were used to immobilize on the surface of the biosensor to enhance overall detection sensitivity. In addition to gold nanoparticles, the magnetic nanoparticles has been demonstrated the applicability for RF DNA detection. The performance of the proposed biosensor was evaluated by the shift of the center frequency of the RF biosensor because the electromagnetic characteristic of the biosensors can be altered by the immobilized multilayer nanoparticles on the biosensor. The experimental results show that the detection limit of the DNA concentration can reach as low as 10 pM, and the largest shift of the center frequency with triple-layer AuNPs and MNPs can approach 0.9 and 0.7 GHz, respectively. Such the achievement implies that the developed biosensor can offer an alternative inexpensive, disposable, and highly sensitive option for application in biomedicine diagnostic systems because the price and size of each biochip can be effectively reduced by using fully polymeric materials and multilayer-detecting structures.     

Preparation of a DNA matrix via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group.

A new methodology for the preparation of addressed DNA matrices is described. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5' end a pyrrole moiety introduced by phosphoramidite chemistry. The electro-controlled synthesis of the copolymer (poly-pyrrole) gives, in one step, a solid conducting film deposited on the surface of an electrode. The resulting polymer consists of pyrrole chains bearing covalently linked oligonucleotide. The polymer growth is limited to the electrode surface, so that it is possible to prepare a DNA matrix on a multiple electrode device by successive copolymerizations. A support bearing four oligonucleotides was used to detect three ras mutations on a synthetic DNA fragment.     

Multilayer DNA/poly(allylamine hydrochloride) microcapsules: assembly and mechanical properties

We report the preparation, characterization, and mechanical properties of DNA/poly(allylamine hydrochloride) (PAH) multilayer microcapsules. The DNA/PAH multilayers were first constructed on a planar support to examine their layer-by-layer buildup. Surface plasmon resonance spectroscopy (SPR) showed a nonlinear growth of the assembly upon each bilayer deposited independently on a concentration of salt. A weak increase in the film thickness with the DNA concentration was, however, detected. A post-treatment of the multilayers in the salt solutions has shown a thinning of the film. The optimal conditions of the planar film growth were used to deposit the same multilayers on the surface of colloidal templates and to study their roughness and morphology with the atomic force microscope (AFM) imaging. When an outer layer is formed by DNA, we observe large domains of oriented parallel DNA loops, while an outer layer formed by PAH shows highly porous morphology. The dissolution of colloidal templates led to a formation of highly porous DNA/PAH microcapsules. We probe their mechanical properties by measuring force-deformation curves with the AFM-related setup. The experiment suggests that the DNA/PAH capsules are softer than capsules made from the flexible polyelectrolytes studied before. The softening is due to both higher permeability and smaller Young's modulus of the shell material. The Young's modulus of the DNA/PAH shells increases after post-treatment in salt solutions of relatively low concentration.     

Gold nanoparticles modified electrode via simple electrografting of in situ generated mercaptophenyl diazonium cations for development of DNA electrochemical biosensor

A novel protocol for development of DNA electrochemical biosensor based on gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE) was proposed, which was carried out by the self-assembly of AuNPs on the mercaptophenyl film (MPF) via simple electrografting of in situ generated mercaptophenyl diazonium cations. The resulting MPF was covalently immobilized on GCE surface via C-C bond with high stability, which was desirable in fabrication of excellent performance biosensors. Probe DNA was self-assembled on AuNPs through the well-known Au-thiol binding. The recognition of fabricated DNA electrochemical biosensor toward complementary single-stranded DNA was determined by differential pulse voltammetry with the use of Co(phen)(3)(3+) as the electrochemical indicator. Taking advantage of amplification effects of AuNPs and stability of MPF, the developed biosensor could detect target DNA with the detection limit of 7.2×10(-11) M, which also exhibits good selectivity, stability and regeneration ability for DNA detection.     

DNA electrochemical biosensor based on thionine-graphene nanocomposite

A novel protocol for development of DNA electrochemical biosensor based on thionine-graphene nanocomposite modified gold electrode was presented. The thionine-graphene nanocomposite layer with highly conductive property was characterized by scanning electron microscopy, transmission electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. An amino-substituted oligonucleotide probe was covalently grafted onto the surface of the thionine-graphene nanocomposite by the cross-linker glutaraldehyde. The hybridization reaction on the modified electrode was monitored by differential pulse voltammetry analysis using an electroactive intercalator daunomycin as the indicator. Under optimum conditions, the proposed biosensor exhibited high sensitivity and low detection limit for detecting complementary oligonucleotide. The complementary oligonucleotide could be quantified in a wide range of 1.0 × 10(-12) to 1.0 × 10(-7)M with a good linearity (R(2)=0.9976) and a low detection limit of 1.26 × 10(-13)M (S/N=3). In addition, the biosensor was highly selective to discriminate one-base or two-base mismatched sequences.     

DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

Here we report a new method to detect DNA point mutations.The method is based on the formation and deformation of double-stranded DNA(dsDNA)membranes on a gold surface.It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface.In these systems,Fe(CN)63- was used as the reporter.As the temperature increases,a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears.At a special temperature,the and single base mutation target.Thus,the system provides a simple and sensitive method to detect DNA point mutations without labeling targets.     

Label-free detection of kanamycin based on the aptamer-functionalized conducting polymer/gold nanocomposite

Highly sensitive label-free detection of kanamycin is achieved with an aptamer sensor based on a conducting polymer/gold self-assembled nanocomposite. The sensor probe is fabricated by covalently immobilizing an in vitro selected DNA aptamer for kanamycin onto gold nanoparticle (AuNP)-comprised conducting polymer, poly-[2, 5-di-(2-thienyl)-1H-pyrrole-1-(p-benzoic acid)] (poly-DPB). The self-assembling of DPB on AuNP is investigated by TEM and UV-vis spectroscopy and the modification of the aptamer sensor is characterized using XPS and electrochemical impedance spectroscopy. The probe is applied to detect kanamycin by using voltammetric techniques. The sensor shows a pair of redox peaks around 0.26/ 0.08 V (vs. Ag/AgCl) for kanamycin captured by the aptamer-immobilized probe. The parameters that can affect the response, such as aptamer concentration, incubation time, temperature, and pH are optimized. The calibration plot shows a linear range from 0.05 μM to 9.0 μM kanamycin with a detection limit of 9.4±0.4 nM. The proposed aptamer sensor is examined with a real sample.     

Nanogold hollow balls with dendritic surface for hybridization of DNA

Au hollow balls are fabricated by adsorption of gold 3.5 nm in diameter onto a mixed vesicle composed of mixed polymerized diacetylene which made of negative charged 10,12-pentacosadiynoic acid (PCDA) and positive charged 10,12-pentacosadiynoic acid 2'-aminoethylamide (PCDA-NH(2)). The morphology of these hollow spheres could be controlled by changing the ratio of PCDA and PCDA-NH(2) and the immobilization and hybridization ability of the gold hollow ball have been investigated using a quartz crystal microbalance (QCM). It was found that a dendritic surface in an appropriate ratio existed. The hybridization amount of target DNA is about three to five times for the Au-mixed hollow ball at an optimal ratio (PCDA/PCDA-NH(2)=1/3) as compared with that for pure Au-PCDA-NH(2), though the immobilization amount of ssDNA on these two samples are almost the same, and the detected limitation of target DNA is extend from 10(-9) to 10(-12) M. The stability against breakage by transportation, combined with the simplicity and efficiency of detection, would offer an important advantage over unpolymerized one. This result shows the possibility to control the morphology and surface of nanogold hollow spheres by changing the ratio of PCDA and PCDA-NH(2) for the develop of a better DNA detection assay, further proving the idea that low surface coverage and higher DNA probe to target DNA ratios lead to optimal hybridization.     

PMMA isocyanate-modified digital discs as a support for oligonucleotide-based assays

A mild chemical procedure for the derivatization of PMMA as the isocyanate on rigid supports is described. The proposal is based on spin-coating the support with a hydroxy-modified PMMA polymer, followed by treatment with (3-isocyanatopropyl)triethoxysilane, developing an isocyanate-ended PMMA. Oligonucleotide hybridization assays performed on this surface demonstrate that the process is simple and highly effective and agree with the results of other modified materials, including aminated PMMA, gold, and glass. As a demonstration, it has been applied to covalently attached aminated oligonucleotides on a modified audio-video compact disc (CD) surface to perform DNA probe hybridization assays. Measurements were carried out with a CD player, by detecting the solid microspots precipitated from an enzymatic reaction (HRP-TMB). This new approach, being a competitive technique, opens a broad horizon of applications for point of care or in situ needs.     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.