Low‐Background,Highly Sensitive DNA Biosensor by Using an Electrically Neutral Cobalt(II) Complex as the Redox Hybridization Indicator

An electrically neutral cobalt complex, [Co(GA)₂(phen)] (GA=glycollic acid, phen=1,10‐phenathroline), was synthesized and its interactions with double‐stranded DNA (dsDNA) were studied by using electrochemical methods on a glassy carbon electrode (GCE). We found that [Co(GA)₂(phen)] could intercalate into the DNA duplex through the planar phen ligand with a high binding constant of 6.2(±0.2)×10⁵ M ^?1. Surface studies showed that the cobalt complex could electrochemically accumulate within the modified dsDNA layer, rather than within the single‐stranded DNA (ssDNA) layer. Based on this feature, the complex was applied as a redox‐active hybridization indicator to detect 18‐base oligonucleotides from the CaMV35S promoter gene. This biosensor presented a very low background signal during hybridization detection and could realize the detection over a wide kinetic range from 1.0×10^?14 M to 1.0×10^?8 M , with a low detection limit of 2.0 fM towards the target sequences. The hybridization selectivity experiments further revealed that the complementary sequence, the one‐base‐mismatched sequence, and the non‐complementary sequence could be well‐distinguished by the cobalt‐complex‐based biosensor.     

Tris(2,2′-bipyridyl)cobalt(III)-doped silica nanoparticle DNA probe for the electrochemical detection of DNA hybridization

A new and sensitive electrochemical DNA hybridization detection assay, using tris(2,2′-bipyridyl)cobalt(III) [Co(bpy)₃³⁺]-doped silica nanoparticles as the oligonucleotide (ODN) labeling tag, and based on voltammetric detection of Co(bpy)₃³⁺ inside silica nanoparticles, is described. Electro-active Co(bpy)₃³⁺ is not possible for directly linking with DNA, it is doped into the silica nanoparticles in the process of nanoparticles synthesis for DNA labeling with trimethoxysilylpropydiethylenetriamine (DETA) and glutaraldehyde as linking agents. The Co(bpy)₃³⁺ labeled DNA probe is used to hybridize with target DNA immobilized on the surface of glassy carbon electrode. Only the complementary sequence DNA (cDNA) could form a double-stranded DNA (dsDNA) with the DNA probe labeled with Co(bpy)₃³⁺ and give an obvious electrochemical response. A three-base mismatch sequence and non-complementary sequence had negligible response. Due to the large number of Co(bpy)₃³⁺ molecules inside silica nanoparticles linked to oligonucleotide DNA probe, the assay showed a high sensitivity. It allows the detection at levels as low as 2.0×10⁻¹⁰ mol l⁻¹ of the target oligonucleotides.     

Naphthalene Diimide Carrying Two Cysteine Termini at Both Imide Linkers as a Molecular Staple

Naphthalene diimide ( 1 ) carrying cysteines at the termini of amide substituents were synthesized to act as a molecular staple of double stranded DNA. Since 1 is able to bind to double stranded DNA with threading intercalation, the complex of 1 with double stranded DNA can be topologically immobilized on a gold surface through the S? Au linkage as confirmed by cyclic voltammetric experiment. Ferrocenyl‐double stranded 23‐mertic oligonucleotide, dsFcODN, was immobilized on gold electrode with 1.0×10¹² molecules cm^?2 when electrode was treated with 2.0 µM dsFcODN and 4.0 µM 1 for 1 h at room temperature. The coverage density was similar to that obtained for the terminal thiol‐modified oligonucleotide. Compound 1 was applied to detect the 321‐meric PCR product of P. gingivalis, which is important in the diagnosis of periodontal disease. This experiment, coupled with the use of ferrocenylnaphthalene diimide, FND as electrochemical indicator for double stranded DNA, resulted in quantitative detection of PCR product within the range of 10 pg µL^?1–10 ng µL^?1 (15 nM–15 µM). The 1 and FND established a simple and rapid detection method of double stranded PCR product with a detection limit of 10 pg µL^?1 (15 nM).     

DNA对Ru(bpy)_2(dppx)~(2+)-SDS体系共振光散射的猝灭作用及其应用

研究了Ru(bpy)2(dppx)2+-SDS-DNA(bpy=2,2′-联吡啶,dppx=7,8-二甲基-吡啶并[3,2-a:2′,3′-c]吩嗪)体系的共振光散射光谱。结果表明,在阴离子表面活性剂十二烷基硫酸钠(SDS)预胶束聚集体存在下,Ru(bpy)2(dppx)2+-SDS体系具有很强的共振光散射,DNA的加入使其共振散射光猝灭。探讨了反应机理。基于DNA对Ru(bpy)2(dppx)2+-SDS体系共振光散射的猝灭作用,建立了共振光散射法测定DNA的新方法。在最佳实验条件下,体系在393nm处的共振光散射猝灭程度与DNA的浓度呈线性关系,线性范围为0.01～1.2mg/L,检出限为1.5μg/L。     

Study on Triplex DNA by Use of Molecular “Light Switch“ Complex of Ru(phen)2(dppx)^2

A new method for the study of triplex DNA is established according the fluorescence enhancement of molecular “Light Switch“ complex of Ru(phen)2(dppx)^2 when it intercalate into triplex DNA.Because the fluorescence intensity of Ru(phen)2(dppx)^2 bonded to triplex DNA is in ths case higher than that bonded to duplex DNA in certain range of DNA concentration,the method is much more sensitive than other methods reported previously.     

A Novel Method to Determine DNA by Use of Molecular “Light Switch” of Ru(phen)2(dppz)

A novel fluorimetric method was developed for selective determination of DNA with the molecular “light switch” complex of Ru(phen)₂(dppz)²⁺. The maximum fluorescence intensity was produced in the pH range 9.3–11.5, with the maximum excitation and emission wavelength of 453.0 and 598.0 nm, respectively. Under the optimum conditions, the fluorescence intensity was in proportion to the concentration of DNA. The linear range for calfthymus DNA, salmon sperm DNA, and herring sperm DNA are 0–0.9 μg/mL. The limits of detection for calfthymus DNA, salmon sperm DNA, and herring sperm DNA are 2.0, 1.8, and 5.4 ng/mL, separately. When the proposed method was used to determine DNA in the presence of some coexisting substances, a satisfactory result was obtained.     

Molecular modeling on the recognition of DNA sequence and conformational repair of sheared DNA by novel chiral metal complex D,L-[Co(phen)2hpip]3+

A study on the recognition of DNA sequence and conformational repair of sheared DNA by Novel Chiral Metal complex D,L-[Co(phen)₂hpip]³⁺ (phen=1,10 phenanthroline, hpip=2-[2-hydroxyphenyl] imidazole [4,5-f][1,10] phenanthroline) is carried out with molecular simulations. The results reveal that two isomers of the complex could both recognize the normal DNA in the minor groove orientation, while recognize the sheared DNA in the major groove orientation and both isomers could convert the conformation of mismatched bases from sheared form to parallel form. Further analysis shows that the steric details of complex’s intercalation to base stack determine the results of recognition, which is induced by the steric collision among ancillary ligand phen, bases and DNA backbone, and by the steric crowding occurring in the process of structural expansion of bases and DNA backbone. Detailed analysis reveals that the conformational repair of mismatched bases relates not only to the steric interactions, but also the π-π stack among normal bases, mismatched bases and hpip ligand.     

Co(phen)33+与6-巯基嘌呤及DNA间相互作用的研究

自1969年首次报道顺铂(cis-[Pt(NH 3 ) 2 Cl 2 ])对肿瘤有强烈抑制作用以来,金属配合物抗肿瘤药物的研究倍受重视 [1,2] ?很多抗肿瘤药物治疗恶性肿瘤疾病都是通过切割人体肿瘤细胞的DNA来实现的?DNA与外源性分子相互作用的研究构成肿瘤形成的机理和一些抗肿瘤药物作用机理的基     

Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> gene

A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 ^INK4A gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 ^INK4A gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.     

Molecular modeling on the recognition of DNA sequence and conformational repair of sheared DNA by novel chiral metal complex D,L-[Co(phen)2hpip]3+

A study on the recognition of DNA sequence and conformational repair of sheared DNA by Novel Chiral Metal complex D,L-[Co(phen)₂hpip]³⁺ (phen=1,10 phenanthroline, hpip=2-[2-hydroxyphenyl] imidazole [4,5-f][1,10] phenanthroline) is carried out with molecular simulations. The results reveal that two isomers of the complex could both recognize the normal DNA in the minor groove orientation, while recognize the sheared DNA in the major groove orientation and both isomers could convert the conformation of mismatched bases from sheared form to parallel form. Further analysis shows that the steric details of complex’s intercalation to base stack determine the results of recognition, which is induced by the steric collision among ancillary ligand phen, bases and DNA backbone, and by the steric crowding occurring in the process of structural expansion of bases and DNA backbone. Detailed analysis reveals that the conformational repair of mismatched bases relates not only to the steric interactions, but also the π-π stack among normal bases, mismatched bases and hpip ligand.     

Rapid discrimination of single-nucleotide mismatches using a microfluidic device with monolayered beads

A microfluidic device incorporating monolayered beads is developed for the discrimination of single-nucleotide mismatches, based on the differential dissociation kinetics between perfect match (PM) and mismatched (MM) duplexes. The monolayered beads are used as solid support for the immobilization of oligonucleotide probes containing a single-base variation. Target oligonucleotides hybridize to the probes, forming either PM duplexes or MM duplexes containing a single mismatch. Optimization studies show that PM and MM duplexes are easily discriminated based on their dissociation but not hybridization kinetics under an optimized buffer composition of 100 mM NaCl and 50% formamide. Detection of single-nucleotide polymorphism (SNP) using the device is demonstrated within 8 min using four probes containing all the possible single-base variants. The device can easily be modified to integrate multiplexed detection, making high-throughput SNP detection possible.     

Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)₂(dppx)]²⁺ (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)₂(dppx)]²⁺ is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)₂(dppx)]²⁺ through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs–Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.     

Rapid high throughput assay for fluorimetric detection of doxorubicin--application of nucleic acid-dye bioprobe

A double stranded DNA based fluorescence bioprobe for anticancer agent (doxorubicin) detection is described. This method provides a new way for sensitive DNA/drug interaction study by a homogeneous assay. The probe employs the long-wavelength intercalating fluorophore TOTO-3^® (TT3). The anticancer agent, doxorubicin, which interacts with the DNA-TT3 complex, was indirectly measured by the decrease in the fluorescence intensity. Various oligonucleotides with different sequences were examined. Doxorubicin has preference for the oligonucleotide 5′AGCACG3′. Enhanced fluorescence observed for the TT3 intercalation with this oligonucleotide makes the DNA-dye complex a suitable bioprobe for doxorubicin detection by competitive assay. A home-built CCD camera setup was applied along with 384 well plate assay format for high throughput fluorescence imaging. The detection limit can be as low as 25 ng mL⁻¹ with an upper limit of 100 μg mL⁻¹. The recovery test with spiked serum sample shows that this method can be a potential routine method for therapeutic drug monitoring (TDM).     

基于NaYF4:Yb,Er上转换荧光纳米颗粒精准检测DNA

建立了一种利用碱基堆积原理并以上转换纳米粒子荧光作为内参的精准检测DNA的方法。该方法首先利用热分解法制备NaYF_4∶Yb,Er上转换荧光纳米颗粒(upconversion nanoparticles,UCNPs),再通过表面羧基化变性牛血清蛋白修饰后与氨基化探针核酸单链共价偶联,形成上转换荧光标记显示探针。最后再基于碱基堆积原理进行杂交检测。研究结果表明以NaYF_4∶Yb,Er荧光强度为内参,根据FAM/UCNP的强度比来定量检测目标DNA浓度比单一的以报告DNA中FAM荧光强度定量检测目标DNA浓度要更为精准,有效地避免了实验中出现的人为操作和仪器误差。本方法不需要进行扩增,检测底限可达到5 nmol·L~(-1),且在较大的浓度范围内有较好的线性关系,同时该方法也有着良好的特异性,能有效区分单碱基错配序列。     

Label-free oligonucleotide detection method based on a new L-cysteine-dihydroartemisinin complex electroactive indicator

A novel base-mismatched oligonucleotide assay method based on label-free electrochemical biosensor was developed, in which the L-cysteine (Cys)-dihydroartemisinin (DHA) complex was used as a new electroactive indicator. In DNA sensor, Cys-DHA complex was initially formed on electrode surface by cathodic scanning, and target oligonucleotide was conjugated with Cys-terminated DHA indicator through electrostatic interaction under optimal pH. The subsequent sequence assay was responsive to hybridization recognition, which target oligonucleotide was captured by the surface-anchored DNA/Cys-DHA probe. The electrochemical signals of biosensor before and after hybridization were compared basing the measurements of semi-derivative linear scan voltammetry (SDLSV) and electrochemical impedance spectroscopy (EIS). On the basis of signal amplification of electroactive indicator and specific recognition of DNA probe, five target oligonucleotides with different mismatched bases were assayed, and a detection limit reached 0.3 nM. Furthermore, atomic force microscopy (AFM) was used to visually characterize specific recognition spots of biosensor at nanoscale. This study demonstrated a new electroactive molecule-based, biomolecule-involved electroactive indicator and its application in recognition and detection of complementary and base-mismatched oligonucleotide.     

荧光探针Ru(phen)2(dppx)2+测定H1N1禽流感病毒DNA

利用荧光探针Ru(phen)2dppx2+与ssDNA作用时不产生荧光或荧光很弱,而与dsDNA作用时荧光增强的机理,将H1N1禽流感病毒ssDNA与其完全互补ssDNA杂交形成dsDNA实现Ru(phen)2dppx2+对H1N1禽流感病毒DNA特定序列（5’-CTA CCA TGC GAA CAA TTC AAC CGA CAC TGT T-3’）的定量检测。在优化的实验条件下,测定H1N1禽流感病毒 DNA的线性范围为9.3×10-10～7.4×10-8 mol/L,线性关系：y = 3.3829x + 8.3948,R2 =0.9982,检出限为5.3×10-10 mol/L。该方法具有操作简单,检测快速,灵敏度高和选择好等优点。     

手性配合物Λ, Δ-[Ru(phen)2hpip]2+的两种结构对含G:T错配DNA识别的分子力学研究

In this work, the recognition of DNA including G：T mismatched pairs by the two different structures of [Ru（phen）2hpip]^2＋ was firstly studied with molecular modeling respectively. The results revealed that all of the four chiral isomers of the two structures could recognize the mismatched DNA from the minor groove orientation especially and the interaction was enantioselective and sitespecific. The two left isomers were more preferential than the right ones. Especially, the structure Ⅱ which had much lower energy after interacting with DNA was the advantaged structure. Detailed energy analysis indicated that the steric interaction in the process of the complex inserting base stack determined the recognition results and the electrostatic interaction made an effect to some extent.     

Chemiluminescence Method for the Determination of Glutathione in Human Serum Using the Ru(phen)3 2+ – KMnO4 System

A simple, rapid and specific chemiluminescence (CL) method for the determination of glutathione has been developed. The method is based on the enhanced CL of the reaction between Ru(phen)₃ ²⁺ (phen = 1,10-phenanthroline) and KMnO₄ by glutathione in HCl medium. Under the optimum conditions, the response is linearly proportional to the concentration of glutathione between 1.5 × 10⁻⁷ and 1.0 × 10⁻⁵ mol L⁻¹. The dection limit for glutathione (5.8 × 10⁻⁸ mol L⁻¹) is about 10 and 200 times better than those of the spectrophotometric method using Ellman regent and the Lucigenin – CL method, respectively. The final procedure allows the determination of glutathione in human serum with recoveries of 92%–108%. A satisfactory agreement was obtained with a mean relative difference of 2.5% compared to the HPLC method.     

基于NaYF4:Yb,Er上转换荧光纳米颗粒精准检测DNA

建立了一种利用碱基堆积原理并以上转换纳米粒子荧光作为内参的精准检测DNA的方法。该方法首先利用热分解法制备NaYF₄：Yb,Er上转换荧光纳米颗粒（upconversion nanoparticles,UCNPs）,再通过表面羧基化变性牛血清蛋白修饰后与氨基化探针核酸单链共价偶联,形成上转换荧光标记显示探针。最后再基于碱基堆积原理进行杂交检测。研究结果表明以NaYF₄：Yb,Er荧光强度为内参,根据FAM/UCNP的强度比来定量检测目标DNA浓度比单一的以报告DNA中FAM荧光强度定量检测目标DNA浓度要更为精准,有效地避免了实验中出现的人为操作和仪器误差。本方法不需要进行扩增,检测底限可达到5 nmol·L^-1,且在较大的浓度范围内有较好的线性关系,同时该方法也有着良好的特异性,能有效区分单碱基错配序列。