An Overview of Analytical Techniques Employed to Evidence Drug-DNA Interactions. Applications to the Design of Genosensors
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paper_title: Capillary electrophoresis for studying drug-DNA interactions

paper_content:

Abstract The development of new drugs to treat disease by binding directly to DNA offers much promise but is reliant on methods to determine the relative affinity of the putative drug for different DNA sequences. Such methods should ideally be rapid and inexpensive as well as reliable. Use of capillary electrophoresis in simple silica columns offers such a method. The development of systems in which the solvent carries a soluble polymer allows the reliable separation of DNA oligomers, of 12–20 bp in length, which can then be titrated with the ligand in competition experiments. The results obtained are comparable with those obtained by footprinting and give direct graphical output, easily analysed for relative binding affinity.
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paper_title: Capillary affinity gel electrophoresis: new technique for specific recognition of DNA sequence and the mutation detection on DNA.

paper_content:

The present state of studies on capillary affinity gel electrophoresis, which is a new technique for the specific recognition of a target DNA sequence, is reviewed. This article includes the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the sequence-specific recognition of DNA and the detection of mutations in specific genes is illustrated.
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paper_title: Biological fingerprinting analysis of traditional Chinese medicines with targeting ADME/Tox property for screening of bioactive compounds by chromatographic and MS methods.

paper_content:

Traditional Chinese medicines (TCMs) are attracting increased global attention because of their potential to provide novel therapeutic agents based on substantial historical records of efficacy in man. Many strategies have been designed for the screening and selection of bioactive compounds from these complex natural products mixtures. Biological fingerprinting analysis (BFA), based on small molecule-biomacromolecule interactions in complex systems, has been applied to screen the multiple bioactive compounds in natural products. Here we review the chromatographic and MS approaches used for BFA of natural products with targeting absorption, distribution, metabolism, elimination and toxicity (ADME/Tox) properties. Such chromatographic methods cover a wide range of applications including liposome, serum proteins, liver homogenate and DNA profiling. MS methods for the characterization of molecular interactions between natural products and target molecules by ESI and MALDI-TOF MS are also discussed.
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paper_title: Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

paper_content:

The rate of DNA supercoil removal by human topoisomerase IB (TopIB) is slowed down by the presence of the camptothecin class of antitumor drugs. By preventing religation, these drugs also prolong the lifetime of the covalent TopIB–DNA complex. Here, we use magnetic tweezers to measure the rate of supercoil removal by drugbound TopIB at a single DNA sequence in real time. This is accomplished by covalently linking camptothecins to a triple helix-forming oligonucleotide that binds at one location on the DNA molecule monitored. Surprisingly, we find that the DNA dynamics with the TopIB–drug interaction restricted to a single DNA sequence are indistinguishable from the dynamics observed when the TopIB–drug interaction takes place at multiple sites. Specifically, the DNA sequence does not affect the instantaneous supercoil removal rate or the degree to which camptothecins increase the lifetime of the covalent complex. Our data suggest that sequencedependent dynamics need not to be taken into account in efforts to develop novel camptothecins.
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paper_title: Characterization of enantioselective binding of racemic natural tetrahydropalmatine to DNA by chromatographic methods.

paper_content:

Abstract A racemate from natural product, tetrahydropalmatine (THP), was characterized on its enantioselective binding to DNA by the chromatographic methods including microdialysis/HPLC, centrifugal ultrifiltration/HPLC and immobilized DNA affinity chromatography. It was found that its (+)-enantiomer was preferential to binding on B-form duplex DNA including calf thymus DNA, AT and GC sequence oligo DNA, as well as triplex oligo DNA. The binding constants of the THP enantiomers to ct-DNA were determined with the methods of microdialysis/HPLC and frontal affinity chromatography. In addition, the DNA structural preference of either enantiomer was evaluated with the chromatographic methods.
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paper_title: Carboplatin interaction with calf-thymus DNA: A FTIR spectroscopic approach

paper_content:

Abstract Carboplatin is a chemotherapeutic drug, used for the treatment of different types of cancers, particularly solid tumors. Carboplatin, like other platinum containing drugs, exerts its cytotoxic effect through DNA binding via cross-linking. It forms interstrand and intrastrand cross-linking with DNA. Intrastrand cross-linking is dominant and believed to be conferring antitumoral efficacy of the drug. This cross-linking results in alteration of DNA winding and bending, which hampers DNA replication and transcription and finally leads to cell death. In the present work, we studied the interaction of carboplatin with calf-thymus DNA in buffer solution under physiological conditions. Different concentrations of carboplatin were incubated with a constant DNA concentration to form carboplatin–DNA complexes. These complexes were studied with Fourier Transform Infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy to understand the binding modes of carboplatin with DNA and its effect on DNA conformation. The results showed that carboplatin binds to DNA through direct interaction of platin–DNA bases (guanine, thymine, adenine and cytosine), with a small perturbation of phosphate group of DNA backbone, while DNA remains in the B-conformation. DNA aggregation was also observed at higher drug concentrations.
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paper_title: A library of IR bands of nucleic acids in solution.

paper_content:

This review presents a compilation and discussion of infrared (IR) bands characteristic of nucleic acids in various conformations. The entire spectral range 1800-800 cm(-1) relevant for DNA/RNA in aqueous solution has been subdivided into four sections. Each section contains descriptions of bands appearing from group specific parts of nucleic acid structure, such as nucleobase, base-sugar, sugar-phosphate and sugar moiety. The approach allows comparisons of information obtained from one spectral region with another. The IR band library should facilitate detailed and unambiguous assignment of structural changes, ligand binding, etc. in nucleic acids from IR spectra. is aimed at highlighting specific features that are useful for following major changes in nucleic acid structures. also concerns some recent results, where IR spectroscopy has been used to obtain semi-quantitative information on coexisting modes of sugar pucker in oligonucleotides.
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paper_title: High-Resolution NMR of DNA and Drug-DNA Interactions.

paper_content:

The advantage of NMR over most other spectroscopic techniques lies in the ability to gain structural and dynamic information at atomic resolution. Every nucleus with spin gives rise to a signal that is characterized by a number of parameters (chemical shift, J-couplings, relaxation data, and NOE connectivities) that can be used to obtain quite detailed structural information about the molecule under study. They can also be used to determine kinetic properties, for example, the interconversion rates of different conformations of a molecule and the exchange rate of free with bound ligand on a macromolecule. NMR has been widely used to study both static and dynamic aspects of DNA structure and drug-DNA interactions.
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paper_title: Biophysical studies on the base specificity and energetics of the DNA interaction of photoactive dye thionine: spectroscopic and calorimetric approach.

paper_content:

In this study absorbance, fluorescence, circular dichroic spectroscopy, viscosity, thermal melting and calorimetric techniques were employed to understand the binding of the phenothiazinium dye, thionine, with deoxyribonucleic acids of varying base composition. Strong hypochromic and bathochromic effects and quenching of fluorescence were observed that showed strong binding of thionine to the DNAs. The binding parameters evaluated from Scatchard analysis through McGhee-von Hippel analysis showed that the binding was non-cooperative and affinities of the order of 10(5)M(-)(1). The results of ferrocyanide fluorescence quenching studies and viscosity experiments, taken together suggested the intercalation of thionine while thermal melting, differential scanning calorimetry and circular dichroic studies provided evidence for the thermal stabilization and conformational perturbations associated with the binding. The thermodynamic parameters elucidated through sensitive isothermal titration calorimetric studies suggested that the binding was exothermic, characterized by negative enthalpy and large positive entropy changes and that the non-electrostatic contributions play a significant role for thionine association to DNA. The heat capacity changes obtained from the temperature dependence of enthalpy changes gave negative values suggesting substantial hydrophobic contribution in the DNA binding process of thionine. Further, an observation of enthalpy-entropy compensation in the DNA binding also suggested the involvement of multiplicity of non covalent interactions in the binding process. The base specificity of the complexation and energetics of the interaction of thionine to DNA are obtained for the first time from this study.
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paper_title: Radiative Decay Engineering: Biophysical and Biomedical Applications

paper_content:

Fluorescence spectroscopy is a widely used research tool in biochemistry and molecular biology. Fluorescence has also become the dominant method enabling the revolution in medical diagnostics, DNA sequencing, and genomics. To date all the fluorescence observables, including spectral shifts, anisotropies, quantum yields, and lifetimes, have all been utilized in basic and applied uses of fluorescence. In this forward-looking article we describe a new opportunity in fluorescence, radiative decay engineering (RDE). By RDE we mean modifying the emission of fluorophores or chromophores by increasing or decreasing their radiative decay rates. In most fluorescence experiments the radiative rates are not changed because these rates depend on the extinction coefficient of the fluorophore. This intrinsic rate is not changed by quenching and is only weakly dependent on environmental effects. Spectral changes are usually caused by changes in the nonradiative rates resulting from quenching or resonance energy transfer. These processes affect the emission by providing additional routes for decay of the excited states without emission. In contrast to the relatively constant radiative rates in free solution, it is known that the radiative rates can be modified by placing the fluorophores at suitable distances from metallic surfaces and particles. This Review summarizes results from the physics literature which demonstrate the effects of metallic surfaces, colloids, or islands on increasing or decreasing emissive rates, increasing the quantum yields of low quantum yield chromophores, decreasing the lifetimes, and directing the typically isotropic emission in specific directions. These effects are not due to reflection of the emitted photons, but rather as the result of the fluorophore dipole interacting with free electrons in the metal. These interactions change the intensity and temporal and spatial distribution of the radiation. We describe the unusual effects expected from increases in the radiative rates with reference to intrinsic and extrinsic biochemical fluorophores. For instance, the decreased lifetime can result in an effective increase in photostability. Proximity to nearby metallic surfaces can also increase the local field and modify the rate of excitation. We predict that the appropriate localization of fluorophores near particles can result in usefully high emission from "nonfluorescent" molecules and million-fold increases in the number of photons observable from each fluorophore. We also describe how RDE can be applied to medical testing and biotechnology. As one example we predict that nearby metal surfaces can be used to increase the low intrinsic quantum yields of nucleic acids and make unlabeled DNA detectable using its intrinsic metal-enhanced fluorescence.
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paper_title: Study on the interaction between rivanol and DNA and its application to DNA assay

paper_content:

Abstract Rivanol (RVN) binds to the double helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data. Extensive hypochromism and red shifts in the absorption spectra were observed when RVN binds to calf thymus DNA (CT DNA), which suggested the intercalation mechanism of RVN into DNA bases. Upon binding to DNA, the fluorescence from RVN was efficiently quenched by the DNA bases, with no shifts in the emission maximum. The large increases in the polarization upon binding to CT DNA supported the intercalation of RVN into the helix. Iodide quenching studies showed that the magnitude of Ksv of the free RVN was higher than that of the bound RVN. The results of competitive binding studies showed that RVN can be displaced by ethidium bromide. Thermal denaturation experiments exhibited that the quenching of the fluorescence from RVN by single strand (ssDNA) was smaller than that by double strand (dsDNA). The results of all above further studies also proved the intercalation of RVN into DNA base stack. Quenching of fluorescence from RVN by DNA can be employed for sensitive detection of DNA. The limit of detection for CT DNA was 16 ng ml−1.
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paper_title: Optical, redox, and DNA-binding properties of phenanthridinium chromophores: elucidating the role of the phenyl substituent for fluorescence enhancement of ethidium in the presence of DNA.

paper_content:

The phenanthridinium chromophores 5-ethyl-6-phenylphenanthridinium (1), 5-ethyl-6-methylphenanthridinium (2), 3,8-diamino-5-ethyl-6-methylphenanthridinium (3), and 3,8-diamino-5-ethyl-6-(4-N,N-diethylaminophenyl)phenanthridinium (4) were characterized by their optical and redox properties. All dyes were applied in titration experiments with a random-sequence 17mer DNA duplex and their binding affinities were determined. The results were compared to well-known ethidium bromide (E). In general, this set of data allows the influence of substituents in positions 3, 6, and 8 on the optical properties of E to be elucidated. Especially, compound 4 was used to compare the weak electron-donating character of the phenyl substituent at position 6 of E with the more electron-donating 4-N,N-diethylaminophenyl group. Analysis of all of the measurements revealed two pairs of chromophores. The first pair, consisting of 1 and 2, lacks the amino groups in positions 3 and 8, and, as a result, these dyes exhibit clearly altered optical and electrochemical properties compared with E. In the presence of DNA, a significant fluorescence quenching was observed. Their binding affinity to DNA is reduced by nearly one order of magnitude. The electronic effect of the phenyl group in position 6 on this type of dye is rather small. The properties of the second set, 3 and 4, are similar to E due to the presence of the two strongly electron-donating amino groups in positions 3 and 8. However, in contrast to 1 and 2, the electron-donating character of the substituent in position 6 of 3 and 4 is critical. The binding, as well as the fluorescence enhancement, is clearly related to the electron-donating effect of this substituent. Accordingly, compound 4 shows the strongest binding affinity and the strongest fluorescence enhancement. Quantum chemical calculations reveal a general mechanism related to the twisted intramolecular charge transfer (TICT) model. Accordingly, an increase of the twist angle between the phenyl ring in position 6 and the phenanthridinium core opens a nonradiative channel in the excited state that depends on the electron-donating character of the phenyl group. Access to this channel is hindered upon binding to DNA.
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paper_title: Criteria for the mode of binding of DNA binding agents

paper_content:

Abstract A complete characterization of DNA binding agents requires that their mode of binding to DNA be established. In the absence of high resolution structural data, the mode of binding is, of necessity, usually inferred indirectly from various solution studies. The purpose of this study is to show that only certain methods can be used reliably to infer the DNA binding mode. Comparative fluorescence and hydrodynamic studies using the proven intercalator ethidium and the groove binder Hoechst 33258 are described. The results of our studies show that while fluorescence intensity, polarization, and quenching measurements can detect a binding interaction of the ligand with DNA, none are sensitive indicators of the binding mode. Fluorescence contact energy transfer studies can reliably indicate intercalation, as can viscosity measurements. Our results illustrate reliable criteria that may be used to distinguish intercalation from groove binding in the absence of high resolution structural data.
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paper_title: Fluorescence Interaction and Determination of Calf Thymus DNA with Two Ethidium Derivatives

paper_content:

In this paper, we reported the syntheses and investigation of the modes of binding to DNA of the two new ethidium derivatives containing benzoyl and phenylacetyl groups of both amines at 3-and 8- positions. The interactions between calf thymus DNA (ct-DNA) and the two derivatives, 3,8-dibenzoylamino-5-ethyl-6-phenylphenantridinium cloride (E2) and 3,8-diphenylacetylamino-5-ethyl-6-phenylphenantridinium chloride (E3), were investigated by fluorescence quenching spectra and UV-vis absorption spectra. The Stern-Volmer quenching constants, binding constants, binding sites and the corresponding thermodynamic parameters DeltaH, DeltaS and DeltaG were calculated at different temperatures. The results indicated the formation of E2 and E3-DNA complexes and van der Waals interactions as the predominant intermolecular forces in stabilizing for each complex. In addition, increasing nucleophilicity of the functional groups at 3- and 8- positions exhibited the respectable increment the DNA binding affinities of derivatives. The results of absorption, ionic strength and iodide ion quenching suggested that the interaction mode of E2 and E3 with ct-DNA was intercalative binding. The limit of detection (LOD) of ct-DNA were 7.49 x 10(-8) (n = 4) and 4.18 x 10(-8) mol/l (n = 7) in presence of E2 and E3, respectively.
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paper_title: Groove binding of a styrylcyanine dye to the DNA double helix : the salt effect

paper_content:

Abstract The cationic styryl dye DSMI binds to the double helical DNA with a high affinity. A red shift of the absorption spectrum and hypochromism accompany the binding of DSMI to calf thymus DNA. The fluorescence yields increase dramatically when DSMI binds to DNA, with no shifts in the emission maximum. These spectral changes are in contrast to the behavior observed with many fluorescent intercalators. Groove binding rather than intercalation of the dye was suggested to be the cause of these spectral changes, based on a variety of experimental results. Consistent with groove binding, the fluorescence enhancements were greater with poly(dA—dT) when compared with poly(dG—dC). The addition of salt or a strong electrolyte to the solution releases the DNA-bound dye cations from the groove and causes a decrease in the fluorescence yield. Therefore, when the dye binds to DNA, the fluorescence quenching with anionic quenchers was enhanced dramatically, in contrast to the expected protection of the probe fluorescence by the DNA helix. Binding of DSMI to DNA inhibits the non-radiative deactivation of the excited state, perhaps owing to the restriction imposed by the narrow minor groove of the DNA. The steric constraints imposed on the dye owing to the binding in the groove result in enhanced fluorescence yields and longer fluorescence lifetimes. Since the lifetimes of the DNA-bound dye were still in the nanosecond range, it is very likely that groove binding of the dye permits twisting in the excited state, which may be important in the excited state dynamics of this class of dyes. The large fluorescence enhancements observed when DSMI binds to the helix and the poor fluorescence yield of the dye in the absence of DNA suggests that DSMI should be quite useful in the staining of DNA gels and for the detection of very low concentrations of DNA.
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paper_title: Study of the lumiescence properties of a new series of quinolizinium salts and their interaction with DNA

paper_content:

The spectrofluorimetric characteristic of a new group of benzo- and methyl- quinolizinium salts at room temperature and 77 LK are reported. At room temperature, linear calibration is wide; 10-9 M 9-cyanobenzo [a] phenathro [9,10-g] quinolizinium chloride can be detected in methanolic solution and 10-7 M in aqueous solution. The polynuclear compounds show the most intense luminescence bands, and a significant hypsochromic shift of the fluorenscence emission maximum was observed at 77 K compared with room temperature. For the 2,3-diphenyl derivatives, the presence of a methoxy substituent produces a marked Stoke's shift, because it causes a decrease in the planarity of the molecule. The benzo compounds are similar in structure to the alkaloid coralyne, which has significant antileuikemic activity. The fused planar aromatic compounds are shown to bind with DNA.
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paper_title: Criteria for the mode of binding of DNA binding agents

paper_content:

Abstract A complete characterization of DNA binding agents requires that their mode of binding to DNA be established. In the absence of high resolution structural data, the mode of binding is, of necessity, usually inferred indirectly from various solution studies. The purpose of this study is to show that only certain methods can be used reliably to infer the DNA binding mode. Comparative fluorescence and hydrodynamic studies using the proven intercalator ethidium and the groove binder Hoechst 33258 are described. The results of our studies show that while fluorescence intensity, polarization, and quenching measurements can detect a binding interaction of the ligand with DNA, none are sensitive indicators of the binding mode. Fluorescence contact energy transfer studies can reliably indicate intercalation, as can viscosity measurements. Our results illustrate reliable criteria that may be used to distinguish intercalation from groove binding in the absence of high resolution structural data.
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paper_title: Radiative Decay Engineering: Biophysical and Biomedical Applications

paper_content:

Fluorescence spectroscopy is a widely used research tool in biochemistry and molecular biology. Fluorescence has also become the dominant method enabling the revolution in medical diagnostics, DNA sequencing, and genomics. To date all the fluorescence observables, including spectral shifts, anisotropies, quantum yields, and lifetimes, have all been utilized in basic and applied uses of fluorescence. In this forward-looking article we describe a new opportunity in fluorescence, radiative decay engineering (RDE). By RDE we mean modifying the emission of fluorophores or chromophores by increasing or decreasing their radiative decay rates. In most fluorescence experiments the radiative rates are not changed because these rates depend on the extinction coefficient of the fluorophore. This intrinsic rate is not changed by quenching and is only weakly dependent on environmental effects. Spectral changes are usually caused by changes in the nonradiative rates resulting from quenching or resonance energy transfer. These processes affect the emission by providing additional routes for decay of the excited states without emission. In contrast to the relatively constant radiative rates in free solution, it is known that the radiative rates can be modified by placing the fluorophores at suitable distances from metallic surfaces and particles. This Review summarizes results from the physics literature which demonstrate the effects of metallic surfaces, colloids, or islands on increasing or decreasing emissive rates, increasing the quantum yields of low quantum yield chromophores, decreasing the lifetimes, and directing the typically isotropic emission in specific directions. These effects are not due to reflection of the emitted photons, but rather as the result of the fluorophore dipole interacting with free electrons in the metal. These interactions change the intensity and temporal and spatial distribution of the radiation. We describe the unusual effects expected from increases in the radiative rates with reference to intrinsic and extrinsic biochemical fluorophores. For instance, the decreased lifetime can result in an effective increase in photostability. Proximity to nearby metallic surfaces can also increase the local field and modify the rate of excitation. We predict that the appropriate localization of fluorophores near particles can result in usefully high emission from "nonfluorescent" molecules and million-fold increases in the number of photons observable from each fluorophore. We also describe how RDE can be applied to medical testing and biotechnology. As one example we predict that nearby metal surfaces can be used to increase the low intrinsic quantum yields of nucleic acids and make unlabeled DNA detectable using its intrinsic metal-enhanced fluorescence.
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paper_title: Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy.

paper_content:

Fluorescence spectroscopy is widely used in biological research. Until recently, essentially all fluorescence experiments were performed using optical energy which has radiated to the far-field. By far-field we mean at least several wavelengths from the fluorophore, but propagating far-field radiation is usually detected at larger macroscopic distances from the sample. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. Near-field interactions are those occurring within a wavelength distance of an excited fluorophore. The spectral properties of fluorophores can be dramatically altered by near-field interactions with the electron clouds present in metals. These interactions modify the emission in ways not seen in classical fluorescence experiments. In this review we provide an intuitive description of the complex physics of plasmons and near-field interactions. Additionally, we summarize the recent work on metal-fluorophore interactions and suggest how these effects will result in new classes of experimental procedures, novel probes, bioassays and devices.
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paper_title: Localized surface plasmon-enhanced fluorescence spectroscopy for highly-sensitive real-time detection of DNA hybridization.

paper_content:

Versatile and highly-sensitive detection of DNA hybridization is described using metal nanostructures-enhanced fluorescence (MEF) emission intensity when fluorescently-labeled DNA oligomers are covalently immobilized on a nanometer-thin amorphous silicon-carbon layer capping the metal nanostructures. The MEF structures are formed by thermal deposition of silver, gold or silver/gold thin films on glass surfaces and post-annealing at 500 degrees C. The choice of the metal film allows for tuning the optical properties of the interface. The metallic nanostructures are subsequently coated with an amorphous thin silicon-carbon alloy (a-Si(0.80)C(0.20): H) layer deposited by PECVD. Carboxydecyl groups are attached on these surfaces through hydrosilylation then reacted with amine-terminated single-stranded DNA oligomers, forming a covalent link. The immobilized DNA is hybridized with its complementary strand carrying a fluorescent label. Through optimization of the thickness of the a-Si(0.80)C(0.20): H alloy overlayer and by working close to resonance conditions for plasmon and fluorophore excitation, the hybridization of very dilute oligomers (5 fM) is easily detected, and the hybridization kinetics can be monitored in situ and in real-time.
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paper_title: Interactions of Fluorophores with Iron Nanoparticles: Metal-Enhanced Fluorescence

paper_content:

Different density iron nanoparticulate substrates were fabricated by thermal vapor deposition in order to study the interactions of fluorophores with iron nanoparticles. We observed metal-enhanced fluorescence (MEF), when fluorophores were placed in close proximity, in the near-field, to the iron nanoparticle deposited substrates. There is often a strong net absorption effect caused by the localized enhanced electromagnetic field of the incident excitation field, when the luminophore is placed near the metal. Subsequently,the electromagnetic field distributions around different size Fe nanoparticles were simulated using FDTD, which revealed that the maximum electric field intensity is predicted to occur over the ∼400−550 nm wavelength range. In addition, the decay time of fluorophores was also reduced near the iron substrates, suggesting both an enhanced electric field and a plasmon-coupling component are the mechanisms for fluorescence enhancement, consistent with our laboratory’s current interpretation ...
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paper_title: Label-free DNA detection on nanostructured Ag surfaces.

paper_content:

The dramatic local electric-field enhancement property of Ag nanoparticles was used as the basis to significantly increase the signal output of a novel label-free (or “self-labeled”) fluorescence-based DNA detection system. In response to identical amounts of analyte, nanostructured Ag substrates provided a posthybridization fluorescent sensor response over 10-fold larger than the response from planar Au substrates. Detection performance strongly depended upon the Ag substrate roughness. Consistent with work by others on metal-enhanced fluorescence, fluorescence intensity also depended strongly on the distance between the fluorophore and the Ag substrate surface. Adjusting the surface roughness, amount of the Ag deposited on the surface, and the DNA probe length allowed for production of an optimized response. In addition to constituting a novel label-free DNA sensor, we anticipate that these structures will provide a unique platform for testing concepts in plasmonics.
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paper_title: Surface plasmon resonance imaging for affinity-based biosensors

paper_content:

SPR imaging (SPRi) is at the forefront of optical label-free and real-time detection. It offers the possibility of monitoring hundreds of biological interactions simultaneously and from the binding profiles, allows the estimation of the kinetic parameters of the interactions between the immobilised probes and the ligands in solution. We review the current state of development of SPRi technology and its application including commercially available SPRi instruments. Attention is also given to surface chemistries for biochip functionalisation and suitable approaches to improve sensitivity.
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paper_title: Fiber-Optic Sensors Based on Surface Plasmon Resonance: A Comprehensive Review

paper_content:

Since the introduction of optical fiber technology in the field of sensor based on the technique of surface plasmon resonance (SPR), fiber-optic SPR sensors have witnessed a lot of advancements. This paper reports on the past, present, and future scope of fiber-optic SPR sensors in the field of sensing of different chemical, physical, and biochemical parameters. A detailed mechanism of the SPR technique for sensing purposes has been discussed. Different new techniques and models in this area that have been introduced are discussed in quite a detail. We have tried to put the different advancements in the order of their chronological evolution. The content of the review article may be of great importance for the research community who are to take the field of fiber-optic SPR sensors as its research endeavors.
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paper_title: Footprinting: a method for determining the sequence selectivity, affinity and kinetics of DNA-binding ligands.

paper_content:

Footprinting is a simple method for assessing the sequence selectivity of DNA-binding ligands. The method is based on the ability of the ligand to protect DNA from cleavage at its binding site. This review describes the use of DNase I and hydroxyl radicals, the most commonly used footprinting probes, in footprinting experiments. The success of a footprinting experiment depends on using an appropriate DNA substrate and we describe how these can best be chosen or designed. Although footprinting was originally developed for assessing a ligand's sequence selectivity, it can also be employed to estimate the binding strength (quantitative footprinting) and to assess the association and dissociation rate constants for slow binding reactions.
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paper_title: DNase I Footprinting

paper_content:

Footprinting is a method for determining the sequence selectivity of DNA-binding compounds in which ligands protect DNA from cleavage at their binding sites. Footprinting templates are typically 50-200 base pairs long, and DNase I is the most commonly used nuclease for these experiments. This chapter describes the preparation and labelling of suitable DNA footprinting substrates, the footprinting experiment itself, and the way in which these data can be used to estimate the dissociation constant of the interaction
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paper_title: Trends in DNA biosensors

paper_content:

Abstract Biosensors have witnessed an escalating interest nowadays, both in the research and commercial fields. Deoxyribonucleic acid (DNA) biosensors (genosensors) have been exploited for their inherent physico-chemical stability and suitability to discriminate different organism strains. The main principle of detection among genosensors relies on specific DNA hybridization, directly on the surface of a physical transducer. This review covers the main DNA immobilization techniques reported so far, new micro- and nanotechnological platforms for biosensing and the transduction mechanisms in genosensors. Clinical applications, in particular, demand large-scale and decentralized DNA testing. New schemes for DNA diagnosis include DNA chips and microfluidics, which couples DNA detection with sample pretreatment under in vivo-like hybridization conditions. Higher sensitivity and specificity may arise from nanoengineered structures, like carbon nanotubes (CNTs) and DNA/protein conjugates. A new platform for universal DNA biosensing is also presented, and its implications for the future of molecular diagnosis are argued.
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paper_title: Nanomaterials as Analytical Tools for Genosensors

paper_content:

Nanomaterials are being increasingly used for the development of electrochemical DNA biosensors, due to the unique electrocatalytic properties found in nanoscale materials. They offer excellent prospects for interfacing biological recognition events with electronic signal transduction and for designing a new generation of bioelectronic devices exhibiting novel functions. In particular, nanomaterials such as noble metal nanoparticles (Au, Pt), carbon nanotubes (CNTs), magnetic nanoparticles, quantum dots and metal oxide nanoparticles have been actively investigated for their applications in DNA biosensors, which have become a new interdisciplinary frontier between biological detection and material science. In this article, we address some of the main advances in this field over the past few years, discussing the issues and challenges with the aim of stimulating a broader interest in developing nanomaterial-based biosensors and improving their applications in disease diagnosis and food safety examination.
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paper_title: Interaction of anticancer herbal drug berberine with DNA immobilized on the glassy carbon electrode.

paper_content:

The interaction of anticancer herbal drug berberine with double-strand DNA (dsDNA) and single-strand DNA (ssDNA) in solution, dsDNA immobilized on the glassy carbon electrode prepared by Langmuir-Blodgett technique, were investigated by electrochemical techniques (cyclic voltammetry, differential pulse voltammetry) and UV spectroscopy. The presence of DNA results in a decrease of the currents and a negative shift of the electrode potentials from the DPV curves of berberine, indicating the dominance of electrostatic interactions. The spectroscopy data confirmed that the predominant interaction between berberine and DNA is electrostatic. The binding of berberine with DNA, when analyzed in terms of the cooperative Hill model, yields the binding constant K(a)=2.2(+/-0.2)x10(4) M(-1), corresponding to the dissociation equilibrium constant K(d)=4.6(+/-0.3)x10(-5) M, which in the range of the applied concentrations of DNA (bp) and berberine, and a Hill coefficient m=1.82(+/-0.08) in Britton-Robinson buffer solution (0.05 M, pH 5.72) at T=298 K (25 degrees C). Apparently, at least two molecules of berberine have to bind as a couple to cause, e.g., the "elementary event" of current change. The results are suggestive for further fruitful applications of this anticancer herbal drug and DNA-modified electrodes.
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paper_title: Aligned carbon nanotube thin films for DNA electrochemical sensing

paper_content:

Abstract Carbon nanotubes are interesting materials for DNA electrochemical sensing due to their unique electric properties: high surface area, fast heterogeneous electron transfer, and electrochemical stability. In this work aligned Carbon NanoTube (CNT) thin films were designed and tested as candidate platforms for DNA immobilization and for the development of an electrochemical genosensor. The films were prepared by Chemical Vapor Deposition (CVD) using acetylene and ammonia as precursor gases and nickel particles as catalyst. A preliminary electrochemical characterization was performed using cyclic voltammetry since, so far, these films have been used only for gas sensing. The surfaces were then covalently functionalized with a DNA oligonucleotide probe, complementary to the sequence of the most common inserts in the GMOs: the Promoter 35S. The genosensor format involved the immobilization of the probe onto the sensor surface, the hybridization with the target-sequence and the electrochemical detection of the duplex formation. Careful attention was paid to the probe immobilization conditions in order to minimize the signal due to non-specifically adsorbed sequences. For the detection of the hybridization event both label-free and enzyme-labelled methods were investigated. In case of the enzyme-labelled method a target concentration at nanomolar level can be easily detected, with a linear response from 50 nM to 200 nM, whereas the label-free method showed a linear response between 0.5 μM and 10 μM. The reproducibility was 11% and 20% with the enzyme-labelled method and the label-free method, respectively. The batch-to-batch reproducibility of the different sensors was also evaluated.
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paper_title: Magneto immunoseparation of pathogenic bacteria and electrochemical magneto genosensing of the double-tagged amplicon.

paper_content:

A rapid and sensitive method for the detection of food pathogenic bacteria is reported. In this approach, the bacteria are captured and preconcentrated from food samples with magnetic beads by immunological reaction with the specific antibody against Salmonella. After the lysis of the captured bacteria, further amplification of the genetic material by PCR with a double-tagging set of primers is performed to confirm the identity of the bacteria. Both steps are rapid alternatives to the time-consuming classical selective enrichment and biochemical/serological tests. The double-tagged amplicon is then detected by electrochemical magneto genosensing. The "IMS/double-tagging PCR/m-GEC electrochemical genosensing" approach is used for the first time for the sensitive detection of Salmonella artificially inoculated into skim milk samples. A limit of detection of 1 CFU mL(-1) was obtained in 3.5 h without any pretreatment, in LB broth and in milk diluted 1/10 in LB. If the skim milk is pre-enriched for 6 h, the method is able to feasibly detect as low as 0.04 CFU mL(-1) (1 CFU in 25 g of milk) with a signal-to-background ratio of 20. Moreover, the method is able to clearly distinguish between pathogenic bacteria such as Salmonella and Escherichia coli. The features of this approach are discussed and compared with classical culture methods and PCR-based assay.
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paper_title: Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

paper_content:

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.
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paper_title: Optical technologies for the read out and quality control of DNA and protein microarrays

paper_content:

Microarray formats have become an important tool for parallel (or multiplexed) monitoring of biomolecular interactions. Surface-immobilized probes like oligonucleotides, cDNA, proteins, or antibodies can be used for the screening of their complementary targets, covering different applications like gene or protein expression profiling, analysis of point mutations, or immunodiagnostics. Numerous reviews have appeared on this topic in recent years, documenting the intriguing progress of these miniaturized assay formats. Most of them highlight all aspects of microarray preparation, surface chemistry, and patterning, and try to give a systematic survey of the different kinds of applications of this new technique. This review places the emphasis on optical technologies for microarray analysis. As the fluorescent read out of microarrays is dominating the field, this topic will be the focus of the review. Basic principles of labeling and signal amplification techniques will be introduced. Recent developments in total internal reflection fluorescence, resonance energy transfer assays, and time-resolved imaging are addressed, as well as non-fluorescent imaging methods. Finally, some label-free detection modes are discussed, such as surface plasmon microscopy or ellipsometry, since these are particularly interesting for microarray development and quality control purposes.
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paper_title: A review of fiber-optic biosensors

paper_content:

Fiber-optic biosensors (FOBS) are optical fiber-derived devices which use optical field to measure biological species such as cells, proteins, and DNA. Because of their efficiency, accuracy, low cost, and convenience, FOBS are promising alternatives to traditional immunological methods for biomolecule measurements. Tapered fiber-optic biosensors (TFOBS) are a type of FOBS which rely on special geometries to expose the evanescent field to interact with samples. In order to amplify sensitivity and selectivity, TFOBS are often used with various optical transduction mechanisms such as changes in refractive index, absorption, fluorescence, and Surface Plasmon Resonance. In this review, the basic principles of TFOBS are summarized. Various common geometries for evanescent sensing and the influence of geometric parameters on optical principles are reviewed. Finally, a detailed account of the studies done to date for biomolecules detection using TFOBS will be provided. © 2007 Elsevier B.V. All rights reserved.
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paper_title: Concentration dependence of a thiazole orange derivative that is used to determine nucleic acid hybridization by an optical biosensor

paper_content:

Abstract Fluorescent intercalating and groove binding dyes that associate with double stranded DNA (dsDNA) are used for detection of hybridization in some sensor and biochip designs. It is possible that dye–dye interactions at concentrations of dye that are relevant to biosensor use can lead to unexpected and undesired emission wavelength shifts and fluorescence quenching interactions. A specific example of such a situation is described in this work, and suggests cautions and considerations for development of quantitative biosensors and biochips. It has been reported [I.I. Timcheva, V.A. Maximova, T.G. Deligeorgiev, N.I. Gadjev, R.W. Sbnis, I.G. Ivanov, FEBS Lett. 405 (1997) 141] that 1-hydroxyethyl-4-[(3-methyl-6-methoxybenzothiazole-2-ylidine)methine] quinolium bromide (TOMEHE), a derivative of thiazole orange, may be useful for quantitative determination of DNA hybridization. In this manuscript, we report evaluation of TOMEHE for use as a fluorescent indicator of DNA hybrid formation on a fiber optic genosensor platform. The wavelength of maximum fluorescence emission of TOMEHE associated with immobilized single-stranded polythymidylic acid isocanucleotides (dT 20 ) on the surface of the optical fiber was determined to be 560±3 nm and was not observed to be significantly different from that in the presence of dsDNA, contrary to the initial report (Timcheva et al., 1997). At high dye concentrations, a bathochromic shift in the fluorescence emission intensity maximum of TOMEHE was observed with both single stranded DNA (ssDNA) and dsDNA, and was believed to be the result of self-association of fluorophore. TOMEHE demonstrated a 10-fold greater fluorescence intensity over ethidium bromide (EB) when bound to dsDNA, but also displayed a 2.5-fold increase in fluorescence intensity over EB when associated with ssDNA. To maximize S / N , many biosensor and biochip experiments use dye concentrations that are in large excess in comparison to the quantity of immobilized DNA. The linearity of fluorescence intensity response for TOMEHE when bound to dsDNA was poor ( r 2 =0.566) over the range studied (1:60 to 1:1 dye:base pair ratios), but improved at dye:base pair ratio ranges below 1:12.
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Title: An Overview of Analytical Techniques Employed to Evidence Drug-DNA Interactions. Applications to the Design of Genosensors

Section 1: Introduction
Description 1: Write about the importance of sensitive analytical techniques in demonstrating drug-DNA interactions, technological advancements, and the application of these techniques in various devices for detection and analysis.

Section 2: Chemical structure of DNA
Description 2: Describe the structure of DNA, its importance in understanding drug-DNA interactions, and present the relevant figures that illustrate DNA structure.

Section 3: A brief summary of the modes of drug-DNA interaction
Description 3: Summarize the different types of interactions between drugs and DNA, including non-covalent and covalent interactions, and discuss their stability and recognition mechanisms.

Section 4: Analytical techniques to evidence drug-DNA interactions
Description 4: Provide an overview of various analytical techniques used to study drug-DNA interactions, including UV-vis spectrophotometry, gel mobility electrophoresis, HPLC-MS, and capillary electrophoresis-mass spectrometry.

Section 5: Vibrational spectroscopy: IR and Raman spectrometries
Description 5: Discuss the use of IR and Raman spectroscopy for analyzing DNA structure and drug-DNA interactions, including the regions of interest and the advantages of these methods.

Section 6: NMR spectroscopy
Description 6: Explain the application of NMR spectroscopy in studying drug-DNA interactions, detailing the parameters observed and the types of nuclei analyzed.

Section 7: UV-VIS absorption spectroscopy
Description 7: Describe how UV-VIS absorption spectroscopy is used to detect drug-DNA interactions, including changes in absorption properties and melting temperature analysis.

Section 8: Circular and linear dichroism
Description 8: Describe circular and linear dichroism techniques and how they provide structural information about drug-DNA complexes and their electronic spectroscopic behavior.

Section 9: Fluorescence emission spectroscopy
Description 9: Discuss the application of fluorescence emission spectroscopy to determine drug-DNA interactions, including fluorescence anisotropy, FRET, and polarization measurements.

Section 10: Metal enhanced fluorescence (MEF)
Description 10: Introduce MEF and its advantages over conventional fluorescence measurements, discussing the principles, mechanisms, and applications in DNA detection.

Section 11: Surface plasmon resonance (SPR)-based techniques
Description 11: Explain SPR-based techniques, how they measure changes in mass on functionalized metal layers due to molecular interactions, and their applications in studying drug-DNA interactions.

Section 12: Enzimatic methods: footprinting
Description 12: Describe the footprinting method to identify sequence-specific DNA-binding by drugs, including the use of DNases and hydroxyl radicals as cleavage agents.

Section 13: Genosensors
Description 13: Define genosensors and describe different types, emphasizing optical and electrochemical genosensors, their mechanisms, and applications in detecting specific DNA sequences.

Section 14: Electrochemical genosensors
Description 14: Provide an overview of electrochemical genosensors, their working principles, types of measurements, and strategies for immobilizing DNA on electrodes.

Section 15: Optical genosensors
Description 15: Explain the technology behind optical genosensors, particularly microarrays and their sensitivity, advantages, and applications in detecting DNA sequences.

Section 16: Conclusion
Description 16: Summarize the importance and applications of studying drug-DNA interactions, the potential for designing new DNA ligands, and the future applications of these analytical techniques and genosensors.

Section 17: Acknowledgements
Description 17: Mention the financial support received from various institutions and the authors’ gratitude towards them.
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