Dye YOYO-1 Research Articles

Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA

The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.

Novel Fluorescent Dyes for Single DNA Molecule Techniques

To answer the demands of scientific and medical imaging issues, the family of nucleic acid fluorescent dyes is constantly enlarging. Most of the developed dyes reveal high qualities in bulk solution assays but are inefficient to produce a strong and sufficiently stable signal to enable the application of single-molecule techniques. Therefore, we tested 12 novel monomeric and homodimeric cyanine dyes for potential single DNA molecule imaging. Although their qualities in bulk solutions have already been described, nothing was known about their behavior on a single-molecule level. All 12 dyes demonstrated strong emission when intercalated into single DNA molecules and stretched on a silanized surface, which makes them the perfect choice for fluorescent microscopy imaging. A comparison of their fluorescence intensity and photostability with the most applicable dyes in single-molecule techniques, fluorescent dyes YOYO-1 and POPO-3, was carried out. They all exhibited a strong signal, comparable to that of YOYO-1. However, in contrast to YOYO-1, which is visualized under a green filter only, their emission permits red filter visualization. As their photostability highly exceeds that of similar spectrum POPO-3 dye, the studied dyes stand out as the best choice for a broad range of solid surface single-molecule applications when yellow to red DNA backbone fluorescence is needed.

Fully Streched Single DNA Molecules in a Nanofluidic Chip Show Large-Scale Structural Variation

When stretching and imaging DNA molecules in nanofluidic devices, it is important to know the relation between the physical length as measured in the lab and the distance along the contour of the DNA. Here a single DNA molecule longer than 1 Mbp is loaded into a nanofluidic device consisting of two crossing nanoslits (85nm x 50 microns) connected to microchannels. An applied pressure creates a stagnation point at the crossing of the nanoslits. The drag force from the fluid stretches the DNA. We determine the degree of stretching of the molecule (i) without the use of markers, (ii) without knowing the contour length of the DNA, and (iii) without having the full DNA molecule inside the field-of-view. The analysis is based on the transverse motion of the DNA due its Brownian motion, i.e. the DNA's response to the thermal fluctuations of the liquid surrounding it. The parameter values obtained by fitting agree well with values we obtain from simplified modeling of the DNA as a cylinder in a parallel flow.Secondly, DNA molecules stained with the intercalating dye YOYO-1 are de- and renatured locally following a modified version of the protocol used in Ref. 1. The result is a melting pattern which reflects the local AT/GC-content. Single molecules are loaded into the chip and imaged. Due to the almost complete stretching of the DNA, structural variations in the size range from kbp to Mbp can be detected and quantified from the melting pattern alone.[1] W. Reisner, N. B. Larsen, A. Silahtaroglu, A. Kristensen, N. Tommerup, J. O. Tegenfeldt, and H. Flyvbjerg, PNAS, 107, 30 (2010) 13294.

Assessing How Cationic Intercalators Affect DNA using Nanofluidic Channels

It has long been known that the commonly used fluorescent dye YOYO-1 (YOYO) stains DNA unevenly at sub-stoichiometric concentrations, and that equilibration is slow. We use this uneven staining to increase the resolution in measurements on how YOYO affects the properties of DNA confined in nanofluidic channels. The emission intensity from each DNA-YOYO complex correlates directly with the amount of YOYO bound. We can thus, in one single experiment, study DNA-YOYO molecules ranging from fully saturated to molecules with a low degree of YOYO bound. Interestingly we find that the extension of the DNA does not increase linearly with increasing amount of YOYO bound. Our data instead suggests that there are two different phases with different behavior. We explain this as being due to a transition from a region where intramolecular electrostatics dominates to where also the charges on the channel walls become important.Our observations are of importance not only for YOYO but are general for cationic ligands that bind to DNA by intercalation and the nanochannels are excellently suited for probing such subtle changes in the physics of the DNA-ligand complex.Finally we show how a mix of YOYO and netropsin, a DNA-binding molecule with extreme selectivity for AT-DNA, can be used to map DNA sequences. When adding the two molecules to DNA simultaneously and stretching it in the nanochannel we obtain an emission intensity variation along the DNA that corresponds to the underlying AT/GC ratio. The intensity variation correlates well with the known sequence and we believe that such maps can be useful for example for fast identification of bacterial infections from extremely small sample volumes.

DNA-intercalator interactions: structural and physical analysis using atomic force microscopy in solution

DNA has a strong affinity for many heterocyclic aromatic dyes, such as acridine and its derivatives. Lerman in 1961 first proposed intercalation as the source of this affinity, and this mode of DNA binding has since attracted considerable research scrutiny. DNA intercalators (molecules that intercalate between DNA base pairs) have attracted particular attention due to their antitumoral activity and because they are widely used to fluorescently label DNA. The interaction between double-stranded DNA and bis-intercalators, such as the fluorescent dye YOYO-1 (dimer of oxazole yellow), has been widely studied. In the literature, there are contradicting data regarding the structure and the rigidity of the complex DNA–YOYO-1. Here, we address this problem using high-resolution atomic force microscopy in solution. We directly measured important structural parameters, such as the helical pitch and the elongation of the complex. By measuring intercalator-induced DNA elongation at different YOYO-1 concentrations, we determined the binding constant. We showed that intercalation induces distinct changes in the molecular elasticity compared to the free double stranded DNA. We found a decrease of the persistence length of DNA with increasing amount of bound YOYO-1, which contrasts with some previous assumptions. This study helps in understanding the physicochemical properties of bis-intercalators as well as the mechanism by which they interact with DNA and this technique can ultimately be applied to a large range of other DNA binding molecules such as anti-cancer or anti-viral drugs.

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.

It's not just leukocytes in cytometry

most common objects of investigation for cytometricanalysis, may it be image or flow cytometry, are leukocytesand cells or cell constituents isolated from cells or tissues.However, there are more than just leukocytes to be looked atin the world of cytometry.Erythrocytes, as the transport vehicles for oxygen, are ofcentral relevance in several red blood cell-related diseases. Thisincludes anemia resulting from various acquired or congenitaldiseases, erythrocytosis, and erythrocyte deformation in sicklecell anemia or spherocytosis, among others. These diseasesmay lead to altered erythrocyte count, reduced hemoglobincontent, shape alterations, or alterations of the cells’ elasticity.All of these changes can be analyzed quantitatively on the sin-gle erythrocyte level using the cytometry approach. Possibly,the most advanced and challenging methods are those thattake advantage of novel optical techniques and perform cyto-metry in vivo (1). By innovative in vivo flow cytometry tech-nology, many of the abovementioned erythrocyte characteris-tics can be obtained (2) but it is still a glimpse into the futurefor most researchers. The image cytometry-based technologydeveloped by Tomaiuolo and colleagues from Napoli, Italy(this issue, page 1040) is presently more accessible. Theauthors compared two flow-based image cytometry methodsto measure the morphology of a large number of erythrocytesflowing through a large (50 lm, unbound) or small (4.7–10lm, confined) capillary and images being captured by ahigh-speed camera. The authors report that their techniqueprovides results comparable to standard technologies such asCoulter Counter with regard to erythrocyte volume, area andvolume distribution. In addition, it renders additionalinformation not available by standard methods such as defor-mation in small capillaries or sphericity index.Philipp and colleagues from Kiel, Germany (this issue,page 1048) addressed the question of how to isolate viableMalaria parasite (Plasmodium falciparum) infected erythro-cytes by fluorescence activated cell sorting. In the past, manyresearchers developed flow cytometry methods to analyze dif-ferent stages of development of Plasmodium spec. within theerythrocyte. The majority of these methods rely on stainingthe DNA of the parasite by a DNA-specific fluorochrome andas an example, the dye YOYO-1 can clearly distinguish differ-ent stages of development (3). However, not all of these dyesare suitable for life sorting because labeling, among others,may need erythrocyte permeability. Philipp and colleaguestested three different DNA dyes and reported that VybrantDyeCycle Violet, a fluorochrome specific to the violet spectralrange, provides best results with regard to sensitive detectionand viable sorting of erythrocytes harboring immature para-sites. Test results for in vitro maturation and reinfection rate ofthese sorted early developmenta l stage parasites is identical tothat of unsorted controls. This new improved method providesan important tool for the investigation of P. falciparum matura-tion and for therapeutic drug development to fight malaria.In bacteriology, a small but scientifically strong and activecytometry community is pushing the limits to improve theanalysis and functional characterizationof mixed bacterial com-munities (seeasexample: Ref. 4). In clinical reality, mixedinfec-tions by different bacterial strains are the usual situation. R€ugerand colleagues from Magdeburg and K€othen, Germany (thisissue, page 1055) raised the questionregarding how to discrimi-

Viral Quantitative Capillary Electrophoresis for Counting and Quality Control of RNA Viruses

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze-thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10(8) to 10(13) ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation.

Quantification of dye-mediated photodamage during single-molecule DNA imaging

Single-molecule fluorescence imaging of DNA-binding proteins has enabled detailed investigations of their interactions. However, the intercalating dyes used to visually locate DNA molecules have the undesirable effect of photochemically damaging the DNA through radical intermediaries. Unfortunately, this damage occurs as single-strand breaks (SSBs), which are visually undetectable but can heavily influence protein behavior. We investigated the formation of SSBs on DNA molecules by the dye YOYO-1 using complementary single-molecule imaging and gel electrophoresis-based damage assays. The single-molecule assay imaged hydrodynamically elongated lambda DNA, enabling the real-time detection of double-strand breaks (DSBs). The gel assay, which used supercoiled plasmid DNA, was sensitive to both SSBs and DSBs. This enabled the quantification of SSBs that precede DSB formation. Using the parameters determined from the gel damage assay, we applied a model of stochastic DNA damage to the time-resolved DNA breakage data, extracting the rates of single-strand breakage at two dye staining ratios and measuring the damage reduction from the radical scavengers ascorbic acid and β-mercaptoethanol. These results enable the estimation of the number of SSBs that occur during imaging and are scalable over a wide range of laser intensities used in fluorescence microscopy.

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)(2)], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)(2)] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100mA beam current of 0.5-μW power varied from 0.6h(-1) for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)(2)] and high salt poly[(dA-dT)(2)]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds.

Viral Quantitative Capillary Electrophoresis for Counting Intact Viruses

The quantification of a virus plays an important role in vaccine development, clinical diagnostics, and environmental contamination assays. In all these cases, it is essential to calculate the concentration or number of intact virus particles (ivp) and estimate the degree of degradation and contamination of virus samples. In this work, we propose a cost-efficient, robust method for the quantification and characterization of intact viruses based on capillary zone electrophoresis. This separation method is demonstrated on vaccinia virus (VV) with oncolytic properties. After virus sample preparation, the solution contains intact VV as well as broken viruses and residual DNA from the host cell used for preparation. Regulatory requirements limit the amount of the host cell DNA that can be present in vaccines or human therapeutics. We apply capillary electrophoresis to separate intact virus particles and the residual DNA and to measure the level of virus contamination with DNA impurities. Intercalating YOYO-1 dye is used to detect the encapsulated and free DNA by laser-induced fluorescence. After soft lysis of VV with proteinase K, all encapsulated DNA is dissolved to the free DNA. The change in peak areas and a DNA calibration curve help determine the initial concentration of intact viruses. This viral quantitative capillary electrophoresis (Viral qCE) is able to quantify the oncolytic vaccinia virus in the range of 10(6) to 10(12) ivp/mL.

Native and fluorescent dye-dependent single-DNA molecule microchip dynamics as measured by differential interference contrast microscopy

In a free solution of 10.0 mM Gly-Gly (pH 8.2), the flow directions of native-DNA and DNA molecules intercalated with the fluorescent dye YOYO-1 were reversed in the microchip channel. These results clearly showed that in a confined space and under specific environmental conditions, the fluorescent dye modified the original properties and behavior of the native-DNA molecule.

DNA analysis by single molecule stretching in nanofluidic biochips

Stretching single DNA molecules by confinement in nanofluidic channels has attracted a great interest during the last few years as a DNA analysis tool. We have designed and fabricated a sealed micro/nanofluidic device for DNA stretching applications, based on the use of the high throughput NanoImprint Lithography (NIL) technology combined with a conventional anodic bonding of the silicon base and Pyrex cover. Using this chip, we have performed single molecule imaging on a bench-top fluorescent microscope system. Lambda phage DNA was used as a model sample to characterize the chip. Single molecules of λ-DNA stained with the fluorescent dye YOYO-1 were stretched in the nanochannel array and the experimental results were analysed to determine the extension factor of the DNA in the chip and the geometrical average of the nanochannel inner diameter. The determination of the extension ratio of the chip provides a method to determining DNA size. The results of this work prove that the developed fabrication process is a good alternative for the fabrication of single molecule DNA biochips and it allows developing a variety of innovative bio/chemical sensors based on single-molecule DNA sequencing devices.

Direct Quantification of Single-Molecules of MicroRNA by Total Internal Reflection Fluorescence Microscopy

MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT-PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis.

Photoswitching of monomeric and dimeric DNA-intercalating cyanine dyes for super-resolution microscopy applications

A growing trend in far-field super resolution fluorescence microscopy based on single molecule photoswitching involves the replacement of photoactivatable fluorophores by common organic dyes in which photoswitching reactions or blinking can be induced. This alternative strategy can greatly simplify the sample preparation and imaging scheme in some cases, and enables its application to a wider range of biological systems. This methodology has been applied successfully to unveil the nanoscale organisation of proteins, but little progress has been seen to date in DNA super-resolution imaging. Previous results have shown that blinking can be induced in the DNA-intercalating dimeric dye YOYO-1 in combination with a reducing buffer, and in turn super-resolution images of DNA can be reconstructed. However, monomeric intercalating dyes like YO-PRO-1 are more advantageous for biological applications. This paper shows that both YO-PRO-1 and YOYO-1 can be used in super-resolution imaging, and different sample preparation strategies are compared in terms of spatial resolution and homogeneity of the reconstructed super-resolution images. Moreover, ensemble and single-molecule experiments provide insight into the switching mechanism. The dyes YOYO-1 and YO-PRO-1 hold great potential for their use in nanoscale imaging of DNA topology in biology and nanoscience.

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

Quantitative measurement of Plasmodium‐infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO‐16 fluorescence

Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (> or =0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD(scidbeta2m-/-) engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparum-infected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation.

Effects of electrostatic screening on the conformation of single DNA molecules confined in a nanochannel

Single T4-DNA molecules were confined in rectangular-shaped channels with a depth of 300 nm and a width in the range of 150-300 nm casted in a poly(dimethylsiloxane) nanofluidic chip. The extensions of the DNA molecules were measured with fluorescence microscopy as a function of the ionic strength and composition of the buffer as well as the DNA intercalation level by the YOYO-1 dye. The data were interpreted with the scaling theory for a wormlike polymer in good solvent, including the effects of confinement, charge, and self-avoidance. It was found that the elongation of the DNA molecules with decreasing ionic strength can be interpreted in terms of an increase of the persistence length. Self-avoidance effects on the extension are moderate, due to the small correlation length imposed by the channel cross-sectional diameter. Intercalation of the dye results in an increase of the DNA contour length and a partial neutralization of the DNA charge, but besides effects of electrostatic origin it has no significant effect on the bare bending rigidity. In the presence of divalent cations, the DNA molecules were observed to contract, but they do not collapse into a condensed structure. It is proposed that this contraction results from a divalent counterion mediated attractive force between the segments of the DNA molecule.

맞물린 나노전극을 가지는 마이크로 캔틸레버의 제작 및 순환전압전류방법을 이용한 DNA의 선택적인 고정화

We present a novel microcantilever device with nano-interdigitated electrodes (nano-IDEs) and DNA selective immobilization on the nano-IDEs for biosensing applications. Using the nano-IDEs and cyclic voltammetric methods, we have achieved selective immobilization of DNA with submicrometer spatial resolution on a freestanding microcantilever. 70~500 ㎚-wide gold (Au) nano-IDEs are fabricated on a lowstress SiNx microcantilever with dimensions of 100~600 ㎛ in length, and 15~60 ㎛ in width, with a 0.5 ㎛ thickness using electron beam lithography and bulk micromachining. Streptavidin is selectively deposited on one side of the nano-IDEs using cyclic voltammetry at a scan rate of 0.1 V/s with a range of -0.2~0.7 V during 1~5 cycles. The selective deposition of dsDNA is confirmed by fluorescence microscopy after labeling with YOYO-1 dye.