Detection Of ssDNA Research Articles

Highly Sensitive Detection of Target ssDNA Based on SERS Liquid Chip Using Suspended Magnetic Nanospheres as Capturing Substrates

A new approach for sensitive detection of a specific ssDNA (single-stranded DNA) sequence based on the surface enhanced Raman spectroscopy (SERS) liquid chip is demonstrated. In this method, the probe DNA (targeting to one part of target ssDNA) was attached to the nano-SERS-tags (poly(styrene-co-acrylic acid)/(silver nanoparticles)/silica composite nanospheres), and the capture DNA (targeting to the other part of target ssDNA) was attached to the Fe3O4/poly(acrylic acid) core/shell nanospheres. The nano-SERS-tags with probe DNA were first allowed to undergo hybridization with the target ssDNA in solution to achieve the best efficiency. Subsequently, the magnetic composite nanospheres with capture DNA were added as the capturing substrates of the target ssDNA combined with the nano-SERS-tags. Upon attraction with an external magnet, the nanospheres (including the nano-SERS-tags) were deposited together due to the hybridization, and the deposit sediment was then analyzed by SERS. Quantitative detection of target ssDNA was achieved based on the well-defined linear correlation between the SERS signal intensity and the target ssDNA quantity in the range of 10 nM to 10 pM, and the limit of detection was approximately 10 pM. Multiplexed detection of up to three different ssDNA targets in one sample was demonstrated using three different types of nano-SERS-tags under a single excitation laser. The experimental results indicated that the liquid-phase DNA sequencing method, thus named the SERS liquid chip (SLC) method, holds significant promises for specific detection of trace targets of organisms.

(Invited) Direct Electrochemical Detection of ssDNA with a Micro Three-Electrode System

The intrinsic electrochemical activity of single strand deoxyribonucleic acid (ssDNA) is investigated with a micro three-electrode system (MTES) integrated inside an in situ electrochemical detection μPCR (EC-μPCR) chip. Based on the MTES, electrochemical cyclic voltammetry (CV) was performed with both normal ssDNA and special ssDNA(Guanine absence) solutions. Experimental results showed that the MTES can directly detect the electrochemical oxidation of the guanine in ssDNA. After a prepolarization step with a positive potential (+500 mV) applied to the MTES for 15 minutes, the electrochemical oxidation peak current of normal ssDNA can be increased about 40%. The detection limit of the MTES with CV method for normal ssDNA is down to 45 nM. The results reveal that label-free and real-time detection of PCR amplicons by directly monitoring the electrochemical oxidation of ssDNA is promising.

Gender and strain-specific differences in the development of steatosis in rats

Non-alcoholic fatty liver disease (NAFLD) is a common problem with a wide variety of phenotypes. While its pathogenesis is still not fully understood, several risk factors for disease progression have been identified. Therefore, defining adequate animal models may serve to unreveal the pathogenesis in NAFLD. We studied Lewis and Sprague-Dawley rats of both genders (n = 6) fed standard (Std) or high-fat (HF) diet for three weeks. Disease stage was assessed by haematoxylin-eosin, Azan Heidenheim and Oil-Red staining, apoptosis by single-stranded DNA (ssDNA) detection and liver regeneration by Ki-67 staining. Serum markers of liver injury and lipid metabolism including adipocytokines were analysed. Livers of both strains and genders fed with HF diet demonstrated evidence of steatosis. Lewis rats developed microvesicular steatosis whereas Sprague-Dawley rats presented macrovesicular steatosis accompanied by pronounced fibrosis. Female gender of both strains was associated with lower steatosis grade and higher proliferation rate (P < 0.05). Gender-specific differences were most prominent in Lewis rats on a HF diet, where females showed lower alkaline phosphatase, cholesterol, triglyceride and leptin levels and a more favourable low-density lipoprotein/high-density lipoprotein ratio than males (P < 0.05). Reverse transcriptase-polymerase chain reaction analysis was performed to demonstrate changes in expression of various genes important for liver regeneration, fibrosis and steatosis. HF diet induced downregulation of proangiogenic genes such as vascular endothelial growth factor receptor 1 and 2 (P < 0.05) in males was not present in females. In conclusion, strain and gender served major roles in disease progression. These differences should be considered when designing studies and may offer new ways to advance therapeutic strategies.

Nucleic acid isolation and enrichment on a microchip

This paper presents a microchip that isolates and enriches target-binding single-stranded DNA (ssDNA) from a randomized DNA mixture using a combination of solid-phase extraction and electrophoresis. Strands of ssDNA in a randomized mixture are captured via specific binding onto target-functionalized microbeads in a microchamber. The strands are further separated from impurities and enriched on-chip via electrophoresis. The microchip consists of two microchambers that are connected by a channel filled with agarose gel. In the isolation chamber, beads functionalized with human immunoglobulin E (IgE) are retained by a weir structure. An integrated heater elevates the temperature in the chamber to elute desired ssDNA from the beads, and electrophoretic transport of the DNA through the gel to the second chamber is accomplished by applying an electric potential difference between the two chambers. Experimental results show that ssDNA expressing binding affinity to IgE was captured and enriched from a sample of ssDNA with random sequences, demonstrating the potential of the microchip to enhance the sensitivity of ssDNA detection methods in dilute and complex biological samples.

Highly-sensitive and label-free indium phosphide biosensor for early phytopathogen diagnosis

The development of highly-sensitive and label-free operating semiconductor-based, biomaterial detecting sensors has important applications in areas such as environmental science, biomedical research and medical diagnostics. In the present study, we developed an Indium Phosphide (InP) semiconductor-based resistive biosensor using the change of its electronic properties upon biomaterial adsorption as sensing element. To detect biomaterial at low concentrations, the procedure of functionalization and covalent biomolecule immobilization was also optimized to guarantee high molecule density and high reproducibility which are prerequisite for reliable results. The characterization, such as biomolecular conjugation efficiency, detection concentration limits, receptor:ligand specificity and concentration detection range was analyzed by using three different biological systems: i) synthetic dsDNA and two phytopathogenic diseases, ii) the severe CB-form of Citrus Tristeza Virus (CTV) and iii) Xylella fastidiosa, both causing great economic loss worldwide. The experimental results show a sensitivity of 1pM for specific ssDNA detection and about 2nM for the specific detection of surface proteins of CTV and X. fastidiosa phytopathogens. A brief comparison with other semiconductor based biosensors and other methodological approaches is discussed and confirms the high sensitivity and reproducibility of our InP based biosensor which could be suitable for reliable early infection diagnosis in environmental and life sciences.

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method—single-stranded DNA (ssDNA) sequencing (SSDS)—that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.

Detection of Target ssDNA Using a Microfabricated Hall Magnetometer with Correlated Optical Readout

Sensing biological agents at the genomic level, while enhancing the response time for biodetection over commonly used, optics-based techniques such as nucleic acid microarrays or enzyme-linked immunosorbent assays (ELISAs), is an important criterion for new biosensors. Here, we describe the successful detection of a 35-base, single-strand nucleic acid target by Hall-based magnetic transduction as a mimic for pathogenic DNA target detection. The detection platform has low background, large signal amplification following target binding and can discriminate a single, 350 nm superparamagnetic bead labeled with DNA. Detection of the target sequence was demonstrated at 364 pM (<2 target DNA strands per bead) target DNA in the presence of 36 μM nontarget (noncomplementary) DNA (<10 ppm target DNA) using optical microscopy detection on a GaAs Hall mimic. The use of Hall magnetometers as magnetic transduction biosensors holds promise for multiplexing applications that can greatly improve point-of-care (POC) diagnostics and subsequent medical care.

Syntheses, optical properties, and bioapplications of the aggregation-induced emission of 2,3,4,5-tetraphenylcyclopenta-2,4-dienyl benzene derivatives

Syntheses of 2,3,4,5-tetraphenylcyclopenta-2,4-dienyl benzene derivatives tailored with seven substituents of various lengths and molecular composition were accomplished and their UV-Vis and fluorescence properties investigated. All of the novel fluorescent dyes displayed varying degrees of aggregation-induced emission (AIE) in the blue region of ∼460 nm in various organic and aqueous solvents. Interestingly, the studies revealed the novel ability of this family of molecules to show strong luminescence in both organic solvents (e.g., %Φf = 69.0% in toluene) and aqueous solutions (e.g., %Φf = 14.0% in H2O) without the need to mix organic and aqueous solvents. Furthermore, fluorescence properties as a function of temperature indicated that both aggregation-induced behavior and the initial degree of solubility played an important role in the overall observed properties of the fluorescent dyes. The importance of this family of molecules for nucleic acid detection and bioimaging applications was demonstrated via the “turn off” detection of cDNA and ss-DNA and by successful tagging and imaging of lens epithelial cells. Taken together, these findings are of great importance for they provide a platform for further rational design and synthesis of luminescent (due to aggregation-induced) and biocompatible (non-toxic) nanoparticles for a wide range of imaging and sensory applications.

Graphene oxide modified light addressable potentiometric sensor and its application for ssDNA monitoring

A light addressable potentiometric sensor (LAPS) is a kind of silicon based semiconductor sensor, and surface modification is a fundamental problem for its application in biological fields. Graphene oxide (GO) based biochemically activated LAPS were proposed, called GO-LAPS. The GO-LAPS were applied to monitoring single strand DNA (ssDNA) probe immobilization and its hybridization with complementary ssDNA molecules of different chain lengths (30, 21 and 14 base pairs, respectively). It was discovered that the curves of LAPS' currents versus analyte concentrations for ssDNA probe binding and the target ssDNA hybridization were different. Explanations were proposed based on the semiconductor's surface-electric-field-effect and the electrical properties of ssDNA molecule. Moreover, comparisons between GO-LAPS and LAPS without GO modification were carried out. Enhanced response currents of GO-LAPS were reported experimentally and analyzed theoretically based on X-ray photoelectron spectroscopy (XPS) of GO-LAPS. The limitation of target ssDNA monitoring was 1 pM to 10 nM, which suggested that this LAPS based platform could be developed as a sensitive means for short chain ssDNA detection.

Replication Stress-Induced Chromosome Breakage Is Correlated with Replication Fork Progression and Is Preceded by Single-Stranded DNA Formation

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or “collapsed” replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with Surface Plasmon Resonance Imaging Measurements

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm(2) area; 45 nm thickness), each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18 000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.

Role of angiotensin-1 receptor blockade in cirrhotic liver resection

The regeneration capacity of cirrhotic livers might be affected by angiotensin-1 (AT1) receptors located on hepatic stellate cells (HSC). The effect of AT1 receptor blockade on microcirculation, fibrosis and liver regeneration was investigated. In 112 Lewis rats, cirrhosis was induced by repetitive intraperitoneal injections of CCl(4) . Six hours, 3, 7 and 14 days after partial hepatectomy or sham operation, rats were sacrificed for analysis. Animals were treated with either vehicle or 5 mg/kg body weight losartan pre-operatively and once daily after surgery by gavage. Microcirculation and portal vein flow were investigated at 6 h. The degree of cirrhosis was assessed by Azan Heidenhein staining, activation of HSC by desmin staining, apoptosis by ssDNA detection and liver regeneration by Ki-67 staining. Changes in expression of various genes important for liver regeneration and fibrosis were analysed at 6 h and 3 days. Haemodynamic parameters and liver enzymes were monitored. Losartan treatment increased sinusoidal diameter, sinusoidal blood flow and portal vein flow after partial hepatectomy (P<0.05), but not after sham operation. AT1 receptor blockade resulted in increased apoptosis early after resection. HSC activation was reduced and after 7 days, a significantly lower degree of cirrhosis in resected animals was observed. Losartan increased the proliferation of hepatocytes at late time-points and of non-parenchymal cells early after partial hepatectomy (P<0.05). Tumour necrosis factor (TNF)-α was significantly upregulated at 6 h and stem cell growth factor (SCF) was downregulated at 3 days (P<0.05). Losartan increased hepatic blood flow, reduced HSC activation and liver fibrosis, but interfered with hepatocyte proliferation after partial hepatectomy in cirrhotic livers.

Somatic Hypermutation In Stereotyped Subset 4 BCRs/mAbs of CLL Patients, Expressing IGHV4-34 gene, Edit Anti-DNA Reactivity

AbstractAbstract 2444Extensive analysis of immunoglobulin gene sequences from chronic lymphocytic leukemia (CLL) patients reveals that ~30% carry stereotyped B-cell antigen receptors (BCRs) with highly homologous HCDR3s, over-represented in selected IGHV/IGHD/IGHJ rearrangements involving such IGHVs as 1–69, 4–34, and 3–21. This finding along with other structurally unique features of CLL BCRS suggests that antigens or superantigens or both may play an active role in the disease.The mutational status of IGHV genes is one of the most powerful prognostic molecular markers in CLL. Patients with IGHV genes with less than 2% differences from the germ line (“unmutated”; U-CLL) have a more aggressive clinical course compared to those with more than 2% differences (“mutated”; M-CLL). In subsets of sequences with stereotyped HCDR3s certain amino acid changes appear to be “CLL-biased”. One of the most distinctive SHM patterns has been described in sequences expressing stereotyped IGHV4-34 BCRs belonging to Subset 4, that are also characterized by long HCDR3s enriched in basic residues (especially arginine).The IGHV4-34 gene encodes antibodies which are autoreactive due to their recognition of the N-acetyllactosamine (NAL) antigenic determinant of the I/i blood group antigen, also expressed on a 220-kDa CD45 B cell-specific isoform; this recognition is mediated in a superantigenic fashion via a motif in HFR1 of the IGHV4-34 sequence. In addition, IGHV4-34 antibodies with HCDR3 sequence motifs enriched in basic (electropositive) amino acids have been shown to react against B cells and DNA, although HCDR3 electropositivity per se does not universally endow IGHV4-34 B cells with this property.Along these lines, we wanted to investigate if the modifications of subset 4 IGHV4-34 by SHM in precursors of the CLL clones could have affected the binding properties if the respective IG receptors, especially against B cells and DNA. We therefore recombinantly expressed 4 mAbs with IGHV4-34 rearrangements, from our CLL patients sample library; mAbs CLL183, CLL240, CLL342 belong to stereotypic subset 4 and have mutated heavy chain immunoglobulin genes, whereas CLL141 expresses an unmutated gene without belonging to any described subset.As previously reported, we tested reactivity of these mAbs with apoptotic and viable B and T cells. All 4 mAbs reacted significantly only with viable cells; the 3 mAbs belonging to subset 4 bound only viable B cells, with a preference for naïve B cells. We also tested the reactivity of these mAbs with single stranded DNA (ssDNA) and double stranded (dsDNA). We used common assays for identifying nuclear autoantigens in autoimmune disorders: 1) for binding total anti-nuclear antigens, human epithelial cell line HEp-2, and 2) for binding dsDNA, Crithidia Luciliae (CL). None of these tests had a strong positive result.To identify a role played by SHM in editing the anti-DNA reactivity, we chose to revert to germline 9 amino acid positions mutated in the IGHV and 1 in the IGKV sequences of CLL240. We either made revertant antibodies retromutated at single positions or at all mutated positions (completely unmutated and germline-like). Enzyme-linked assay (ELISA) for the semi-quantitative detection of ssDNA and dsDNA showed that the complete revertant antibody regained the ability to strongly bind ssDNA and partially dsDNA. The light chain did not appear to play a crucial role in the reacquisition of the binding, because the recombinant mAb expressing only the IGKV in the germline sequence did not bind ssDNA or dsDNA, as did the original recombinant mAb. Some of the revertants also showed a stronger binding to normal human B cells, with a few of them acquiring a partial ability to also bind T cells. The role of the single revertants is being further analyzed.Few IGHV4-34 CLL sequences are altered in the specific HFR1 motif responsible for NAL binding. This suggests that these IGHV4-34 expressing CLL cells could still bind and be stimulated for clonal expansion by NAL epitopes present in self and exogenous antigens. However, the findings of the present study also indicate that the distinctive modifications introduced by SHM in the stereotyped IGHV4-34 BCRs of subset 4 CLL likely represent a censoring mechanism for avoiding intense self-reactivity, mediated by the arginine-rich HCDR3s, and subsequent clonal deletion. Disclosures:No relevant conflicts of interest to declare.

Single-strand DNA detection using a planar photonic-crystal-waveguide-based sensor

We report an experimental demonstration of single-strand DNA (ssDNA) detection at room temperature using a photonic-crystal-waveguide-based optical sensor. The sensor surface was previously biofunctionalized with ssDNA probes to be used as specific target receptors. Our experiments showed that it is possible to detect these hybridization events using planar photonic-crystal structures, reaching an estimated detection limit as low as 19.8 nM for the detection of the complementary DNA strand.

Prognostic significance of apoptotic cell death in bladder cancer: a tissue microarray study on 179 urothelial carcinomas from cystectomy specimens

To assess the prognostic significance of apoptosis related markers in bladder cancer. A tissue microarray containing 179 bladder carcinomas from cystectomy specimens was analysed immunohistochemically for active caspase-3, single-stranded DNA (ssDNA), p53, Bcl-2, Bax, and COX-2, in correlation to clinicopathological factors. Active caspase-3, ssDNA, p53, Bax and COX-2 were more frequently observed among high grade and higher stage (> or =T2) carcinomas compared with low grade and lower stage (T1) tumours. On the contrary, Bcl-2 was more frequently detected in T1 than in > or =T2 carcinomas. Active caspase-3 correlated with a better survival of the patients. The decreased detection of active caspase-3 and ssDNA and the increased presence of Bcl-2 in T1 carcinomas suggest that alterations in interrelated apoptosis markers may play an important role in the progression of urothelial carcinoma from a superficially infiltrating to a muscle invading tumour and would help to better characterise a subpopulation of T1 carcinomas that could profit from early cystectomy or more aggressive adjuvant chemotherapy. Active caspase-3 might be an important prognostic factor in bladder cancer.

Direct electrochemistry of thermally denatured calf thymus DNA on a poly(methyl methacrylate)–graphite microcomposite electrode

Remarkable direct electrochemical behaviors of thermally denatured calf thymus DNA (ssDNA) on the poly(methyl methacrylate)-graphite powder microcomposite electrode (PMMA/GME) were observed and investigated. The result indicated that the PMMA/GME showed great promotion of the electrochemical response towards the ssDNA oxidation due to the specific characteristics of the nanostructured interface on the resultant composite electrode. Two irreversible oxidation peaks at +1.04 and +0.76 V with obvious negative movement of oxidation potential and increase of the oxidation current (vs. Ag/AgCl) were observed on the PMMA/GME, which corresponded to the oxidation of adenine and guanine residues in the ssDNA molecules. Therefore, detection of ssDNA was then performed at the PMMA/GME. Under the optimal conditions, a good linear relationship was obtained between the oxidation peak currents of guanine or adenine residues and the ssDNA concentration in the range of 5.9 × 10(-3) to ca. 1.1 mg mL(-1) with a detection limit of 1.0 μg mL(-1). This PMMA/GME exhibits some outstanding advantages, such as ease of fabrication, high stability, renewable surface, excellent stability, mechanical rigidity and high electrochemical reactivity, which holds huge promise for further DNA biosensor design.

Nanoparticle based enhancement of electrochemical DNA hybridization signal using nanoporous electrodes

A novel nanoparticle-based enhanced methodology for the detection of ssDNA using nanoporous alumina filter membranes, containing pores of 200 nm in diameter, is reported. The blockage of the pores due to the hybridization is detected by measuring the decrease in the differential pulse voltammetric response of the [Fe(CN)(6)](4-/3-) redox indicator and using screen-printed carbon electrodes as transducing platform. Furthermore, 20 nm gold nanoparticle (AuNPs) tags are used in order to increase the sensitivity of the assay. The enhancement mechanism of DNA detection is due to an additional blocking effect induced by hybridization reaction by bringing AuNPs inside the pores. The developed methodology can be extended to other biosensing systems with interest not only for DNA but also for proteins and cells. The developed nanochannel/nanoparticle biosensing system would have enormous potential in future miniaturized designs adapted to mass production technologies such as screen-printing technology.

Sensitive DNA biosensor improved by Luteolin copper(II) as indicator based on silver nanoparticles and carbon nanotubes modified electrode

A novel and sensitive electrochemical DNA biosensor has been developed for the detection of DNA hybridization. The biosensor was proposed by using copper(II) complex of Luteolin C 30H 18CuO 12 (CuL 2) as an electroactive indicator based on silver nanoparticles and multi-walled carbon nanotubes (Ag/MWCNTs) modified glassy carbon electrode (GCE). In this method, the 4-aminobenzoic acid (4-ABA) and Ag nanoparticles were covalently grafted on MWCNTs to form Ag/4-ABA/MWCNTs. The proposed method dramatically increased DNA attachment quantity and complementary ssDNA detection sensitivity for its large surface area and good charge-transport characteristics. DNA hybridization detection was performed using CuL 2 as an electroactive indicator. The CuL 2 was synthesized and characterized using elemental analysis (EA) and IR spectroscopy. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction between CuL 2 and ds-oligonucleotides (dsDNA). It was revealed that CuL 2 presented high electrochemical activity on GCE, and it could be intercalated into the double helices of dsDNA. The target ssDNA of the human hepatitis B virus (HBV) was quantified in a linear range from 3.23 × 10 −12 to 5.31 × 10 −9 M ( r = 0.9983). A detection limit of 6.46 × 10 −13 M (3 σ, n = 11) was achieved.

Interactions between cationic conjugated polyelectrolyte and DNA and a label‐free method for DNA detection based on conjugated polyelectrolyte complexes

AbstractThe electrostatic complexes of single‐stranded deoxyribonucleic acid (ssDNA) and a cationic conjugated polyelectrolyte (CPE), poly{9,9‐di[3‐(1‐ethyl‐1,1‐dimethyl ammonio)propyl]‐2,7‐fluorenyl‐alt‐1,4‐phenylene dibromide} (PFN), were investigated. Fluorescence emission of PFN solution (10 μmol/L) can be drastically quenched to about one fourth of its original intensity in the presence of a trace amount (2.6 μmol/L) of ssDNA. The effect of oligonucleotide length on the fluorescence quenching behavior was also investigated. In contrast to single‐stranded DNA with 20 bases (ssDNA‐20), ssDNA with 40 bases (ssDNA‐40) induces a relatively higher quenching efficiency and larger red‐shift of PFN emission maximum. The binding constant of ssDNA‐20 and PFN is estimated to be 1.12 × 1021. At extremely low concentration (10 nmol/L), PFN can respond to 0.2 nmol/L (or 2 × 10−10 mol/L) of ssDNA‐20 by significant enhancement of its emission intensity. The result is contrary to the observation in the relative higher concentration, and its mechanism is postulated. Based on the high binding ability of ssDNA with cationic CPE, a label‐free method for ssDNA detection is designed. It uses an electrostatic complex of cationic PFN and an anionic CPE, which exhibits fluorescence resonance energy transfer (FRET) between the two components. Addition of ssDNA improves the FRET extent, indicated by obvious change of fluorescence spectra of the conjugated polyelectrolyte complex. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Nanoscale film structure related to capacitive effects in ethylene-glycol monolayers

A large improvement of the electrochemical capacitive detection of ssDNA and antigens by using ethylene-glycol alkanethiol monolayers has been recently demonstrated. Alkanethiol monolayers without ethylene-glycol functionalization do not show the required electrochemical stability. In this paper, we demonstrate that both phenomena are related to their morphological structure at nano-scale. Ethylene-glycol monolayers were compared with similar films without ethylene-glycol function. Capacitance measurements were used to verify the improved capacitance stability of the electrochemical interface. AFM images were used to investigate films packing and morphology at the nano-scale. This letter demonstrates that films without ethylene-glycol segment present deep grooves which are related to electrochemical instability while the improvement in case of ethylene-glycols is due to the absence of such a feature.