Amperometric detection of nucleic acid at femtomolar levels with a nucleic acid/electrochemical activator bilayer on gold electrode

Cationic redox polymers containing osmium-bipyridine complexes strongly interact with anionic enzymes, such as glucose oxidase and peroxidases, and electrochemically "activate" the enzymes. On the basis of these observations, attempts were made to develop an ultrasensitive nucleic acid biosensor. A mixed monolayer of single-stranded oligonucleotide capture probe and 16-mercaptohexadecanoic acid was formed on a gold electrode through self-assembly. Following hybridization with a complementary nucleic acid and glucose oxidase labeled oligonucleotide detection probe, a cationic redox polymer (electrochemical activator) overcoating was applied to the electrode through layer-by-layer electrostatic self-assembly. The formation of an anionic-cationic bilayer brought the glucose oxidase in electrical contact with the redox polymer, making the bilayer an electrocatalyst for the oxidation of glucose. Thus, nucleic acid molecules were quantified amperometrically at femtomolar levels. The effect of experimental variables on the amperometric response was investigated and optimized to maximize the sensitivity and speed up the assay time. A detection limit of 1.0 fmol/L in 1.0-microL droplets and a linear current-concentration relationship up to 800 fmol/L were attained following a 30-min hybridization. The biosensor was applied to the detection of the 16S gene in a mixture of Escherichia coli 16S + 32S rRNA and a full-length rat housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), of a RT-PCR product.     

Quantitative method for specific nucleic acid sequences using competitive polymerase chain reaction with an alternately binding probe

We have developed a simple, cost-effective, and accurate method for the quantification of specific nucleic acid sequences by the combined use of competitive PCR and a sequence-specific fluorescent probe that binds to either the gene of interest (target) or internal standard (competitor), referred to as alternately binding probe (ABProbe). In this method, the target and competitor were coamplified with the ABProbe, and then the fluorescence intensity was measured. The ratio of the target to the competitor can be calculated from the fluorescence intensity of the ABProbe using fluorescence quenching and fluorescence resonance energy transfer, that is, the starting quantity of the target is successfully calculated by end-point fluorescence measurement. Therefore, this method eliminates the complex post-PCR steps and expensive devices for real-time fluorescence measurement. We called this method alternately binding probe competitive PCR (ABC-PCR). We quantified amoA as a model target by ABC-PCR and real-time PCR. By comparison, the sensitivity, accuracy, and precision of ABC-PCR were similar to those of real-time PCR. Moreover, ABC-PCR was able to correctly quantify DNA even when PCR was inhibited by humic acid; therefore, this method will enable accurate DNA quantification for biological samples that contain PCR inhibitors.     

Electrochemical biosensors employing an internal electrode attachment site and achieving reversible, high gain detection of specific nucleic acid sequences

Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives), or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ～800% at saturating target, a detection limit of just 50 pM, and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus, these devices are fully reusable.     

Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics

A polymeric microfluidic device for solid-phase extraction (SPE)-based isolation of nucleic acids is demonstrated. The plastic chip can function as a disposable sample preparation system for different biological and diagnostic applications. The chip was fabricated in a cyclic polyolefin by hot-embossing with a master mold. The solid phase consisted of a porous monolithic polymer column impregnated with silica particles. The extraction was achieved due to the binding of nucleic acids to the silica particles in the monolith. The solid phase was formed within the channels of the device by in situ photoinitiated polymerization of a mixture of methacrylate and dimethacrylate monomers, UV-sensitive free-radical initiator, and porogenic solvents. The channel surfaces were pretreated via photografting to covalently attach the monolith to the channel walls. The solid phase prepared by this method allowed for successful extraction and elution of nucleic acids in the polymeric microchip.     

Voltammetric characterization of a N,N'-diphenyl-p-phenylenediamine-loaded screen-printed electrode: a disposable sensor for hydrogen sulfide

The voltammetric response of a 10% (by weight) N,N'-diphenyl-p-phenylenediamine (DPPD) and 90% (by weight) carbon and binder screen-printed electrode has been examined in aqueous media over a range of pH using cyclic voltammetry both in the presence and in the absence of sulfide. In the absence, the screen-printed electrode undergoes an initial oxidative process on the surface of the solid organic particles to form an insoluble layer of the corresponding cation radical salt, DPPD(*)(+)X(-), where X(-) is an anion present in the solution. The charge transfer is thought to occur at the three-phase boundary between solid DPPD, carbon, and the aqueous solution. At higher potentials, a second oxidative wave is observed that is attributed to the oxidation of the bulk DPPD with intercalation of the anion species present to form a solid phase of DPPD(*)(+)X(-). The two voltammetric processes were found to stabilize after repetitive scanning, after which time, sulfide was added to the solution. The voltammetric response was found to respond to sulfide by showing a decrease in both the oxidative and reductive waves, which can be attributed to the sulfide effectively blocking the three-phase boundary. The response was found to be independent of the electrode used and at pH 4 produced a linear range from 20 to 165 microM, and a limit of detection of 7.5 microM for sulfide detection was achieved.     

Stripping analysis of nucleic acids at a heated carbon paste electrode

A new electrically heated carbon paste electrode has been developed for performing adsorptive stripping measurements of trace nucleic acids. Such coupling of electrochemistry at electrically heated electrodes with adsorptive constant-current stripping chronopotentiometry offers distinct advantages for trace measurements of nucleic acids. The application of increased temperatures during the deposition step results in dramatic (4-34-fold, depending on temperature applied) enhancement of the stripping signal. Such improvement is attributed to the accumulation step at the heated electrode. Forced thermal convection near the electrode surface facilitates the use of quiescent solutions and hence of ultrasmall volumes. Using an electrode temperature of 32 degrees C and a quiescent solution during the 1-min accumulation, the response is linear over the 1-8 mg/L range tested, with a detection limit of 0.5 mg/L. Such electrode heating technology offers great promise for various applications involving thermal manipulations of nucleic acids.     

Vibrational coupling as a probe of adsorption at different structural sites on a stepped single-crystal electrode

Adsorption of carbon monoxide at step and terrace sites on a Pt(557) ≡ Pt(s)-[6(111) × (100)] electrode was detected with infrared spectroscopy. Vibrational coupling between adsorbates provided insights into the assembly of molecules at the different structural sites. The intermolecular coupling was weak at low coverages as CO ordered along the steps. For coverages between 40 and 70% of saturation, separate bands assignable to CO on steps and CO on terraces appeared. Coupling across this coverage range was markedly weaker on Pt(557) than on the structurally related Pt(335) ≡ Pt(s)-[4(111) × (100)] electrode surface. The results indicate that, after the steps fill, CO populates the terraces on Pt(557) at random rather than by ordering in alignment with the steps. At coverages below saturation, vibrational bands assignable to CO molecules at step and terrace sites are affected differently by changes in electrode potential. The potential-induced spectral changes for the terrace CO bands are similar to those of Pt(111)/CO, but the step CO bands show deviations from this trend at hydrogen adsorption potentials.     

Functionalization of silica nanoparticles for nucleic acid delivery

Silica nanoparticles (SiNPs) have been widely engineered for biomedical applications, such as bioimaging and drug delivery, because of their high tunability, which allows them to perform specific functions. In this review, we discuss the functionalization and performance of SiNPs for nucleic acid delivery. Nucleic acids, including plasmid DNA (pDNA) and small interfering RNA (siRNA), constitute the next generation molecular drugs for the treatment of intractable diseases. However, their low bioavailability requires delivery systems that can circumvent nuclease attack and kidney filtration to ensure efficient access to the target cell cytoplasm or nucleus. First, we discussed the biological significance of nucleic acids and the parameters required for their successful delivery. Next, we reviewed SiNP designing for nucleic acid delivery with respect to nucleic acid loading and release, cellular uptake, endosomal escape, and biocompatibility. In addition, we discussed the co-delivery potential of SiNPs. Finally, we analyzed the current challenges and future directions of SiNPs for advanced nucleic acid delivery.     

Noncompetitive immunoassay of small analytes at the femtomolar level by affinity probe capillary electrophoresis: direct analysis of digoxin using a uniform-labeled scFv immunoreagent

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Site-directed mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-microm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 degrees C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A "mix-and-inject" assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes.     

Localization and quantification of carbon-centered radicals on any amino acid of a protein

A general strategy to localize and quantify carbon-centered radicals within proteins is described. The methodology was first exemplified on amino acids and then on a peptide. This method is applicable to any protein system regardless of size, and the site of hydrogen abstraction by *OH on all residues within proteins is easily and accurately detected.     

Direct determination of oxalic acid by a bare platinum electrode contrasting a platinum nanoparticles-modified glassy carbon electrode

A bare platinum disk electrode without further decoration was directly used to determine oxalic acid (OA), showing good linear ranges of 0.57–104.01 μM and 104.01–228.75 μM with a low detection limit of 0.38 μM (S/N = 3). In contrast, platinum nanoparticles (PtNPs) dispersed on a glassy carbon electrode were successfully achieved by an one-step electrochemical deposition method, possessing relatively wider linear detection ranges of 1.14–342.80 μM and 342.80–548.92 μM for OA with a lower detection limit of 0.28 μM (S/N = 3). Both the proposed electrochemical sensors exhibit great reproducibility, stability and selectivity. In particular, they have been applied to the determination of OA in real spinach samples, showing excellent analytical performance.     

多孔的修饰电极对抗坏血酸的电催化氧化

通过循环伏安(CV)法制备了聚苯胺/聚砜(PAN/PSF)复合膜修饰电极,考察了电极对抗坏血酸(AA)的电催化性能。结果显示,PAN/PSF复合膜修饰电极对AA有很好的电催化氧化作用和高的稳定性。氧化峰电流与AA浓度在一定浓度范围内呈良好的线性关系,检测限为6×10-6 mol/L。该电极制备简单、灵敏度高、检测限低,对水果中AA具有很好的检测效果。     

Electrooxidation of ascorbic acid on a polyaniline-deposited nickel electrode: surface modification of a non-platinum metal for an electrooxidative analysis

The electrooxidation of ascorbic acid (H2A), which does not occur on a bare Ni electrode, has been shown to take place on a polyaniline (PANI)-coated Ni electrode in aqueous electrolytes of a wide pH range. The characteristic voltammetric peak of PANI in 0.1 M H2SO4 at 0.2 V vs SCE corresponding to the transformation of leucoemeraldine to emeraldine gradually diminishes with an increase in concentration of H2A as a result of adsorption. This peak disappears before the appearance of another peak corresponding to the oxidation of H2A at a concentration of 1 mM. The irreversible oxidation current of H2A exhibits a linear dependence on the concentration. The effect of adsorption of H2A on PANI has been shown to increase the voltammetric peak current. A study on the variation of the PANI thickness and its influence on the voltammetric oxidation of H2A has led to an optimum thickness of 1.6 microm. The oxidation currents on the porous PANI/Ni electrode have been found to be several times higher at lower potentials in comparison with the data of a Pt electrode. The reaction has also been studied by ac impedance spectroscopy. In alkaline electrolytes, the Nyquist impedance plot is characterized by two semicircles instead of a single semicircle in acidic electrolytes. Thus, Ni, which is a non-platinum metal, has been found to be useful, by surface modification with PANI, for electrooxidation of H2A. The data are reproducible in the electrolytes of a wide pH range, thus suggesting a good stability, reusability and a long life for the PANI/Ni electrodes.     

Evaluation of internal standards in a competitive nucleic acid sequence-based amplification assay

An end-point quantitative nucleic acid sequence-based amplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyte E. coli clpB mRNA was developed and statistically analyzed. Electrochemiluminescence was chosen as a highly sensitive detection means allowing careful evaluation of the internal standards used. The two internal standards examined had been designed previously using a novel and rapid NASBA-based method. Initially, each standard was used separately in a NASBA reaction; subsequently, two internal standards were added into one reaction at different concentrations. The accuracy and precision of the data obtained were analyzed using linear and multiple regression analysis. In the case of single-standard reactions, the accuracy was >95% and the precision >98.5%. In the case of double-standard reactions, the accuracy increased to >97%. With a single internal standard, 3 orders of magnitude of target sequence could be quantified; using three different concentrations of one internal standard, the dynamic range increased to 5 orders of magnitude. In both cases, a detection limit as low as 0.14 pg of target sequence was obtained. In the case of double-internal standard reactions, a dynamic range with 5 orders of magnitude and a detection limit of 1.76 pg was determined. The high-performance quality of the internal standards was assumed to be in part due to the unique synthesis process using two NASBA reactions rather than traditional cloning techniques.     

基于核酸检测的虾过敏源标准物质研究

选择最常食用的海产对虾作为虾过敏源标准物质的候选物,提取并纯化其基因组DNA,采用序列测定法对设定的目标基因成分进行定性鉴定。用数字PCR技术精确确定海虾16S基因、虾真核生物18S基因、胡萝卜真核生物18S基因的拷贝数,以胡萝卜基因组DNA为本底,重量法配置虾16S基因拷贝数/真核生物18S基因组拷贝数比值为1%。评定虾16S基因拷贝数/真核生物18S基因拷贝数比值的定值不确定度,扩展不确定度为0.09%。并利用数字PCR仪对配置的标准物质进行特性量值验证,结果一致性良好。可为基于核酸检测的食物过敏源标准物质的研制提供方法参考。     

An immunomagnetic electrochemical sensor based on a perfluorosulfonate-coated screen-printed electrode for the determination of 2,4-dichlorophenoxyacetic acid.

A disposable immunomagnetic electrochemical sensor involving a magnetic particle-based solid phase and a Nafion film-coated screen-printed electrode (Nafion-SPE) stuck at the bottom of a polystyrene cylinder (microwell of 300 microL) was developed and evaluated in a competitive immunoassay of the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The competitive binding of 2,4-D and 2,4-D labled with alkaline phosphatase (AP) for a limited amount of polyclonal anti-2,4-D antibody-coated magnetic beads was monitored electrochemically by measuring the AP labled++ activity bound to the beads. The phosphoric acid ester of [[(4-hydroxyphenyl)amino]-carbonyl]cobaltocenium hexafluorophosphate was used as the AP substrate. This anionic substrate (S-) is enzymatically transformed at pH 9.0 into a cationic phenol derivative (P+) which can be easily accumulated in the polyanionic Nafion coating and determined by cyclic voltammetry. During the enzyme reaction, the AP-associated beads were localized on the surface of the Nafion-SPE with the aid of a magnet, thus effectively increasing the concentration of P+ in the Nafion-modified electrode vicinity. The enzyme generation of P+ close to the electrode surface, and thereby to the Nafion film, resulted in a high amplification of the response. A detection limit of 0.01 microgram L-1 2,4-D was thus achieved. The performance of the sensor was successfully evaluated on river water samples spiked with 2,4-D, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.     

转基因番茄及其核酸检测技术研究进展

番茄作为重要的GMCs/GMF(genetically modified crops/food,转基因作物,食品)之一,在我国已被列入第一批实施标识管理的农业GMOs(genetically modified organisms,转基因生物)目录.通过对全球转基因番茄及其核酸检测技术研究现状的分析,阐述了转基因番茄的主要研究方向是研发具有延熟、抗虫等优良性能的番茄新品系,应用于疫苗以及其他药用性蛋白的生物反应器研究;总结了全球已获准上市和在研的重要转基因番茄品系的基因表达盒信息及其核酸检测研究数据;剖析了在转基因番茄核酸检测技术研究中尚存在的如关键性技术标准化以及标准物质缺乏等问题,并提出研究展望.希望为建立与国际接轨的转基因番茄量值溯源传递关键技术体系提供参考.     

Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer

In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.