电化学DNA生物传感器*

对特异DNA序列的检测在基因相关疾病的诊断、军事反恐和环境监测等方面均具有非常重要的意义,DNA传感器的研究就是为了满足对特异DNA序列的快速、便捷、高灵敏度和高选择性检测的需要。近年来涌现出了多种传感策略,根据检测方法的不同可以大致分为光学传感器、电化学传感器、声学传感器等。由于电化学检测方法本身所具有的灵敏、快速、低成本和低能耗等特点,电化学DNA传感器已成为一个非常活跃的研究领域并在近几年中得到了快速发展。本文概括了近年来在DNA传感器的重要分支——电化学DNA传感器领域内的一些重要进展,主要包括DNA探针在传感界面上的固定方法和各种电化学DNA杂交信号的检测方法。     

稀土及其配合物对核酸的断裂作用

人工核酸酶是一类具有限制性内切酶的功能、能高效高选择性地催化水解DNA 或RNA 的断裂工具。它们一般由核酸结构识别系统及催化断裂系统组成, 将两种功能有效地结合起来, 可模拟核酸的酶切反应。本文综述了稀土及其配合物对核酸的断裂作用, 并对其断裂机制进行了探讨。     

Biomolecular interactions and tools for their recognition: focus on the quartz crystal microbalance and its diverse surface chemistries and applications

Interactions between molecules are ubiquitous and occur in our bodies, the food we eat, the air we breathe, and myriad additional contexts. Although numerous tools are available for the recognition of biomolecular interactions, such tools are often limited in their sensitivity, expensive, and difficult to modify for various uses. In contrast, the quartz crystal microbalance (QCM) has sub-nanogram detection capabilities, is label-free, is inexpensive to create, and can be readily modified with a number of diverse surface chemistries to detect and characterize diverse interactions. To maximize the versatility of the QCM, scientists need to know available methods by which QCM surfaces can be modified. Therefore, in addition to summarizing the various tools currently used for biomolecular recognition, explicating the fundamental principles of the QCM as a tool for biomolecular recognition, and comparing the QCM with other acoustic sensors, we systematically review the numerous types of surface chemistries-including hydrophobic bonds, ionic bonds, hydrogen bonds, self-assembled monolayers, plasma-polymerized films, photochemistry, and sensing ionic liquids-used to functionalize QCMs for various purposes. We also review the QCM's diverse applications, which include the detection of gaseous species, detection of carbohydrates, detection of nucleic acids, detection of non-enzymatic proteins, characterization of enzymatic activity, detection of antigens and antibodies, detection of cells, and detection of drugs. Finally, we discuss the ultimate goals of and potential barriers to the development of future QCMs.     

New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance

The hemoglobin (Hb) released from erythrocytes is a primary nutritive component for many blood-feeding parasites. The aspartic protease cathepsin D is a hemoglobinase that is involved in the Hb degradation process and is considered an interesting target for chemotherapy intervention. However, traditional enzymatic assays for studying Hb degradation utilize spectrophotometric techniques, which do not allow real-time monitoring and can present serious interference problems. Herein, we describe a biosensor using simple approach for the real-time monitoring of Hb hydrolysis as well as an efficient screening method for natural products as enzymatic inhibitors using a quartz crystal microbalance (QCM) technique. Hemoglobin was anchored on the quartz crystal surface using mixed self-assembled monolayers. The addition of the enzyme caused a mass change (frequency shift) due to Hb hydrolysis, which was monitored in real time. From the frequency change patterns of the Hb-functionalized QCM, we evaluated the enzymatic reaction by determining the kinetic parameters of product formation (k_cat). The QCM enzymatic assay using immobilized human Hb was shown to be an excellent approach for screening possible inhibitors in complex mixtures, opening up a new avenue for the discovery of novel inhibitors.     

Hydrolysis of nucleic acids in single-cell protein concentrates using immobilized benzonase

Hydrolysis of nucleic acids for single-cell protein concentrates has been carried out in one step using immobilized benzonase on corn cob. The immobilization is carried out by tosylation of primary alcohols of cellulose of corn cob. The immobilized benzonase is more stable vs pH changes than native benzonase, but the same optimum values of [Mg(II)] and temperature are obtained. The DNase activity is greater than the RNase activity. The percentage of DNA is reduced to 3-6% and that of RNA to 50%. The protein loss is negligible (1%). The enzymatic activity per weight unit of enzyme is greater in the case of benzonase that in reported data for other nucleases insolubilized on corn cob by the same procedure.     

Electrochemical quartz crystal microbalance study of azurin adsorption onto an alkanethiol self-assembled monolayer on gold

A quartz crystal microbalance coupled with electrochemistry was used to examine the adsorption of azurin on a gold electrode modified with a self-assembled monolayer of octanethiol. Azurin adsorbed irreversibly to form a densely packed monolayer. The rate of azurin adsorption was related to the bulk concentration of azurin in solution within the concentration range studied. At a high azurin concentration (2.75 muM), adsorption was rapid with a stable adsorption maximum attained in 2-3 min. At a lower azurin solution concentration (0.35 muM), the time to reach a stable adsorption maximum was approximately 30 min. Interestingly, the maximum surface concentration attained for all solution concentrations studied by the QCM method was 25 +/- 1 pmol cm-2, close to that predicted for monolayer coverage. The dissipation was monitored during adsorption, and only small changes were detected, implying a rigid adsorption model, as needed when using the Sauerbrey equation. Cyclic voltammetric data were consistent with a one-electron, surface-confined CuII/CuI azurin process with fast electron-transfer kinetics. The electroactive surface concentration calculated using voltammetry was 7 +/- 1 pmol cm-2. The differences between the QCM and voltammetrically determined surface coverage values reflect, predominantly, the different measurement methods but imply that all surface-confined azurin is not electrochemically active on the time scale of cyclic voltammetry.     

QCM and EC-SPR Studies of Cytochrome cSelf-assembled on Au Electrode and Enhancement of SPR Signal by Au Nanoparticles

Quartz crystal microbalance(QCM) and cyclic voltammetry(CV) were used to characterize the monolayer of cytochrome c(Cyt c), which was adsorbed on gold film modified with alkanethiol mixed monolayer. A direct comparison of protein surface coverages calculated from QCM and cyclic voltammetric measurements illustrates that the ratio of the electroactive Cyt c to the total surface-confined Cyt cis 34%, which suggests that the orientation is a main factor affecting the electroactivity of Cyt c. Moreover, surface plasmon resonance(SPR) measurement combined with CV “in situ” was used to investigate the conformational change of Cyt c in the redox process. Besides, Au nanoparticles(Au NPs) were adsorbed on the surface of Cyt c. The result indicates that Au NPs promote electron transfer between Cyt c and the gold electrode, and SPR result suggests Au NPs enhance SPR signal.     

Oligonucleotide optical switches for intracellular sensing

Fluorescence imaging coupled with nanotechnology is making possible the development of powerful tools in the biological field for applications such as cellular imaging and intracellular messenger RNA monitoring and detection. The delivery of fluorescent probes into cells and tissues is currently receiving growing interest because such molecules, often coupled to nanodimensional materials, can conveniently allow the preparation of small tools to spy on cellular mechanisms with high specificity and sensitivity. The purpose of this review is to provide an exhaustive overview of current research in oligonucleotide optical switches for intracellular sensing with a focus on the engineering methods adopted for these oligonucleotides and the more recent and fascinating techniques for their internalization into living cells. Oligonucleotide optical switches can be defined as specifically designed short nucleic acid molecules capable of turning on or modifying their light emission on molecular interaction with well-defined molecular targets. Molecular beacons, aptamer beacons, hybrid molecular probes, and simpler linear oligonucleotide switches are the most promising optical nanosensors proposed in recent years. The intracellular targets which have been considered for sensing are a plethora of messenger-RNA-expressing cellular proteins and enzymes, or, directly, proteins or small molecules in the case of sensing through aptamer-based switches. Engineering methods, including modification of the oligonucleotide itself with locked nucleic acids, peptide nucleic acids, or l-DNA nucleotides, have been proposed to enhance the stability of nucleases and to prevent false-negative and high background optical signals. Conventional delivery techniques are treated here together with more innovative methods based on the coupling of the switches with nano-objects.     

Design of a colicin E7 based chimeric zinc-finger nuclease

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.     

New Method Showing the Influence of Matrix Components in Leuconostoc mesenteroides Biofilm Formation

Studying biofilm formation and influence of the matrix composition was heavy because only old and long methods were employed up to now: confocal microscopy, fluorescent chemical markers, and/or dying techniques. In this context, an innovative tool, the BioFilm Ring Test, was here employed to explore the role of exopolysaccharides, proteins, and nucleic acids in the formation of biofilm by Leuconostoc mesenteroides. The principle is to add magnetic particles in the culture medium. When a biofilm is formed, particles are unable to migrate in the media to form a ring when a magnet is brought nearer to the well. Therefore, culture media supplemented with proteases, glycanases, and/or nucleases allowed us to identify the involvement of these substances in L. mesenteroides biofilm formation. The results permitted to demonstrate that dextran, proteins, and nucleic acids are implied in biofilm formation.     

The Synthesis of Oligo- and Polynucleotides

Problems and results of the synthesis of oligonucleotides are reviewed. The central role of the nucleic acids in biochemistry is a challenge to synthesize nucleic acids of known base sequence and chain length. Oligomers with various sequences of up to 12 members and homo-oligomers with a maximum chain length of 30 nucleotides can be obtained by chemical synthesis. The enzymatic synthesis of ribonucleic acids can be directed in such a way that polynucleotides with definite sequences are produced.     

Graphene oxide integrated sensor for electrochemical monitoring of mitomycin C-DNA interaction

We present a graphene oxide (GO) integrated disposable electrochemical sensor for the enhanced detection of nucleic acids and the sensitive monitoring of the surface-confined interactions between the anticancer drug mitomycin C (MC) and DNA. Interfacial interactions between immobilized calf thymus double-stranded (dsDNA) and anticancer drug MC were investigated using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. Based on three repetitive voltammetric measurements of 120 μg mL(-1) DNA immobilized on GO-modified electrodes, the RSD % (n = 3) was calculated as 10.47% and the detection limit (DL) for dsDNA was found to be 9.06 μg mL(-1). EIS studies revealed that the binding of the drug MC to dsDNA leads to a gradual decrease of its negative charge. As a consequence of this interaction, the negative redox species were allowed to approach the electrode, and thus increase the charge transfer kinetics. On the other hand, DPV studies exploited the decrease of the guanine signal due to drug binding as the basis for specifically probing the biointeraction process between MC and dsDNA.     

Carbon-nanotube-modified glassy carbon electrodes for amplified label-free electrochemical detection of DNA hybridization

The preparation and attractive performance of carbon-nanotube modified glassy-carbon (CNT/GC) electrodes for improved detection of purines, nucleic acids, and DNA hybridization are described. The surface-confined multiwall carbon-nanotube (MWCNT) facilitates the adsorptive accumulation of the guanine nucleobase and greatly enhances its oxidation signal. The advantages of CNT/GC electrodes are illustrated from comparison to the common unmodified glassy carbon, carbon paste and graphite pencil electrodes. The dramatic amplification of the guanine signal has been combined with a label-free electrical detection of DNA hybridization. Factors influencing the enhancement of the guanine signal are assessed and optimized. The performance characteristics of the amplified label-free electrochemical detection of DNA hybridization are reported in connection to measurements of nucleic-acid segments related to the breast-cancer BRCA1 gene.     

Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.     

Cytochrome c self-assembly on alkanethiol monolayer electrodes as characterized by AFM, IR, QCM, and direct electrochemistry

With the advantage of carbodiimide coupling chemistry, horse heart cytochrome c (cyt c) has been covalently immobilized onto self-assembled monolayers (SAMs) from 11-mercaptoundecanoic acid (MUDA) developed on single-crystal or polycrystalline gold substrate surfaces. The cyt c immobilized substrates thus prepared have been characterized by atomic force microscopy (AFM); we have succeeded in obtaining surface topographical images down to single-protein resolution. AFM imaging has also shown densely packed, uniform protein monolayer formation that is highly suggestive of self-assembly of cyt c molecules on MUDA SAMs. Covalent attachment of cyt c has been further evidenced by reflection-absorption FT-IR as well as microgravimetric analysis using a quartz crystal microbalance (QCM). In the latter, the specific MUDA and cyt c surface concentrations were determined to be 0.86 +/- 0.11 nmol cm-2 (n = 5) and 28 +/- 12 pmol cm-2 (n = 5), both of which agree fairly well with their theoretical counterparts. The obtained QCM chips having the cyt c/MUDA/Au interfacial structure were found to be capable of the direct electrochemistry of the surface-attached cyt c molecules. Cyclic voltammetric measurements on the chips gave particular redox waves showing characteristics of surface process. The electroactive protein surface concentration was determined to be 7.2 +/- 4.8 pmol cm-2 (n = 6); it was almost consistent with values found in literature, while it was limited to 26% in magnitude for the QCM data. This was deemed to have arisen from the orientation variation of the surface-confined cyt c molecules and is discussed briefly.     

Nucleic acids induced peptide-based AIE nanoparticles for fast cell imaging

Herein, we utilized nucleic acids induced peptide co-assembly strategy to develop novel nucleic acids induced peptide-based AIE (NIP-AIE) nanoparticles. Strong fluorescent of AIE could be observed when a little amount of nucleic acids was added into the peptide solution, and the intensity could be regulated by the concentration of nucleic acids. This AIE nanoparticle with good biocompatibility could achieve fast cell imaging. It is also proved that the fluorescence intensity of AIE decreased with time, which indicates that the reducible cross-linkers of Wpc peptide by GSH and nanoparticles gradually disintegrate in cell. Based on the different of AIE fluorescence signals which regulated by the formation and disintegration of nanoparticles, this AIE system is expected to be used for real-time monitoring of drug release from peptide-based nano carriers in vivo or in vitro, and may provide a new platform for the construction of other organic AIE nanoparticles.     

Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging

Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets’ copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.     

Electrochemical biosensors based on nucleic acids and their use in bioaffinity assays for determining DNA and and its effectors

The analytical capabilities of electrochemical biosensors based on nucleic acids are systematized. Immobilization methods that retain the biological activity of nucleic acids and provide an opportunity to use them as multipurpose analytical reagents are described. The use of the above sensors in bioaffinity assays for determining DNA and its effectors in biochemical analysis and environmental monitoring and for determining the nucleotide composition of DNA is demonstrated in many examples.