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Confinement of single molecules within nanoscale environments is crucial in a range of fields, including biomedicine, genomics, and biophysics. Here, we present a method that can concentrate, confine, and linearly stretch DNA molecules within a single optical field of view using dielectrophoretic (DEP) force. The method can convert an open surface into one confining DNA molecules without a requirement for bonding, hydrodynamic or mechanical components. We use a transverse DEP field between a top coverslip and a bottom substrate, both of which are coated with a transparent conductive material. Both layers are attached using double-sided tape, defining the chamber. The nanofeatures lie at the “floor” and do not require any bonding. With the application of an alternating (AC) electric field (2 V p-p ) between the top and bottom electrodes, a DEP field gradient is established and used to concentrate, confine and linearly extend DNA in nanogrooves as small as 100-nm in width. We also demonstrate reversible loading/unloading of DNA molecules into nanogrooves and nanopits by switching frequency (between 10 kHz to 100 kHz). The technology presented in this paper provides a new method for single-molecule trapping and analysis.

We show that genomic-length DNA molecules imaged in nanochannels have an extension along the channel that scales linearly with the contour length of the polymer, in agreement with the scaling arguments developed by de Gennes for self-avoiding confined polymers. This fundamental relationship allows us to measure directly the contour length of single DNA molecules confined in the channels, and the statistical analysis of the dynamics of the polymer in the nanochannel allows us to compute the SD of the mean of the extension. This statistical analysis allows us to measure the extension of λ DNA multimers with a 130-nm SD in 1 min.

We use molecular dynamics simulation to probe the non-equilibrium physics of two nanochannel-confined semiflexible polymers in a homogeneous flow field. We find that for sufficiently stiff chains the internal organization of the two chains takes the form of interwoven folds and circular coils. This organization can lead to mixing or demixing depending on chain stiffness and flow speed. At low and intermediate flow, the two chains adopt a folded configuration, which favours mixing. At high flow, the two chains adopt a predominantly coiled configuration. The coiled configuration results in demixing when the chains are compressed from an initially demixed condition and mixing when the chains are compressed from an initially mixed condition. We find that the mixing/demixing behaviour is governed by the ratio of the number of folded segments of one chain relative to the other at low flow and by the degree of coiling in both chains at high flow. For decreasing stiffness, the chains start to aggregate locally instead of mixing smoothly at low and intermediate flow. In the limit of completely flexible chains, the two chains either completely segregate at low flow, or adopt a locally demixed configuration consisting of large aggregates of one chain relative to the other that undergo complex stochastic dynamics, diffusing, disintegrating, and reforming at intermediate flow. The transition from complete segregation to the aggregate-dominated configuration occurs when the linear intra-chain ordering breaks down. Graphical abstract

We show that genomic-length DNA molecules imaged in nanochannels have an extension along the channel that scales linearly with the contour length of the polymer, in agreement with the scaling arguments developed by de Gennes for self-avoiding confined polymers. This fundamental relationship allows us to measure directly the contour length of single DNA molecules confined in the channels, and the statistical analysis of the dynamics of the polymer in the nanochannel allows us to compute the SD of the mean of the extension. This statistical analysis allows us to measure the extension of lambda DNA multimers with a 130-nm SD in 1 min.

We use a nanofluidic system to investigate the emergence of thermally driven collective phenomena along a single polymer chain. In our approach, a single DNA molecule is confined in a nanofluidic slit etched with arrays of embedded nanocavities; the cavity lattice is designed so that a single chain occupies multiple cavities. Fluorescent video-microscopy data shows that waves of excess fluorescence propagate across the cavity-straddling molecule, corresponding to propagating fluctuations of contour overdensity in the cavities. The waves are quantified by examining the correlation in intensity fluctuations between neighbouring cavities. Correlations grow from an anti-correlated minimum to a correlated maximum before decaying, corresponding to a transfer of contour between neighbouring cavities at a fixed transfer time-scale. The observed dynamics can be modelled using Langevin dynamics simulations and a minimal lattice model of coupled diffusion. This study shows how confinement-based sculpting of the polymer equilibrium configuration, by renormalizing the physical system into a series of discrete cavity states, can lead to new types of dynamic collective phenomena.

The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. For dsDNA confined in nanochannels below a critical width roughly twice the persistence length, there is a cross-over in the fundamental physics: a transition occurs between a regime dominated by self-exclusion interactions and a regime dominated by bending rigidity. Using a combination of electron beam lithography (EBL) and nano imprint lithography (NIL) channels have been fabricated that vary in width from 400nm down to 30 nm. The cross-over scale is identified by measuring the extension of DNA as a function of width: above the critical width, the stretch versus width curve has a power-law behavior which we identify with the self-exclusion regime. Below the crossover length, we observe a deviation from the self-exclusion power-law consistent with a theory developed by T. Odijk. The Brownian fluctuations of the confined DNA, which are critical for single molecule sequencing techniques, were also measured as a function of channel width. The relaxation time of the fluctuations exhibits a novel non-monotonic width-dependence: the relaxation time is maximized at the cross-over width.

We have performed single-molecule studies of GFP-LacI repressor proteins bound to bacteriophage λ DNA containing a 256 tandem lac operator insertion confined in nanochannels. An integrated photon molecular counting method was developed to determine the number of proteins bound to DNA. By using this method, we determined the saturated mean occupancy of the 256 tandem lac operators to be 13, which constitutes only 2.5% of the available sites. This low occupancy level suggests that the repressors influence each other even when they are widely separated, at distances on the order of 200 nm, or several DNA persistence lengths.

Haptoglobin Polymorphism Predicts 30-Day Mortality and Heart Failure in Patients With Diabetes and Acute Myocardial Infarction Mahmoud Suleiman 1 , Doron Aronson 1 , Rabea Asleh 2 , Michael R. Kapeliovich 1 , Ariel Roguin 1 , Simcha R. Meisel 1 , Michael Shochat 1 , Abeer Sulieman 2 , Shimon A. Reisner 1 , Walter Markiewicz 1 , Haim Hammerman 1 , Rachel Lotan 2 , Nina S. Levy 2 and Andrew P. Levy 2 1 Department of Cardiology, Rambam Medical Center, Haifa, Israel 2 Technion Faculty of Medicine, Haifa, Israel Address correspondence and reprint requests to Andrew P. Levy MD, PhD, FACC, Technion Faculty of Medicine, Rappaport Building, Bat Galim, Haifa, 31096, Israel. E-mail: alevy{at}tx.technion.ac.il Abstract Patients with diabetes presenting with acute myocardial infarction (AMI) have an increased rate of death and heart failure. Patients with diabetes homozygous for the haptoglobin (Hp) 1 allele (Hp 1-1) develop fewer vascular complications. We tested the hypothesis that Hp type is related to the outcome of patients with diabetes presenting with AMI. We prospectively assessed the relationship between Hp type and 30-day mortality and heart failure in 1,437 patients with AMI (506 with diabetes). Multivariate logistic regression identified a significant interaction between Hp type and diabetes status on these outcome measures. Hp type was not related to outcome among patients without diabetes. In contrast, Hp 1-1 was associated with a strong protective effect with regard to the primary end point of death (OR 0.14, P = 0.015) and for death and heart failure (OR 0.35; 95% CI 0.15–0.86, P = 0.018) among patients with diabetes. Finally, among patients with diabetes, Hp 1-1 was associated with smaller infarct size. This study demonstrates that in patients with diabetes and AMI, the Hp type is an important determinant of clinical outcome and infarct size. AMI, acute myocardial infarction Hp, haptoglobin Footnotes M.S. and D.A. contributed equally to this study. A.P.L. has served on the advisory board of, holds stock in, and has received honoraria from Haptoguard. Accepted May 23, 2005. Received February 20, 2005. DIABETES

To evaluate the acute effect of minocycline on the pericardium in the experimental animal and in the human with malignant pericardial disease. A prospective study in open-chest dogs and in humans. Experimental surgery laboratory, medical school; coronary care unit, university hospital. Twenty-three open-chest dogs were divided into four groups according to the solution injected intrapericardially: (1) minocycline, 5 mg/kg; (2) minocycline, 10 mg/kg; (3) normal saline solution, 100 mL, followed by minocycline, 10 mg/kg; (4) a mixture of 50 mL of the dog's own blood mixed ex vivo with minocycline, 10 mg/kg to evaluate the effect of rising pH of minocycline solution. The extent of myocardial injury is evaluated by measuring ST-T segment deviation in six standard bipolar leads and in three unipolar electrograms recorded over the left ventricular pericardial surface. The pH of the various minocycline solutions is measured. Nine consecutive patients with malignant cardiac tamponade receiving minocycline intrapericardially are evaluated for the appearance of chest pain and ECG changes. Minocycline (5 and 10 mg/kg) caused marked, transient ST-T segment deviation in all dogs, whether or not saline solution was previously injected into the pericardial sac. Prior mixing of minocycline with blood markedly increased the acidic pH of the minocycline solution and significantly reduced the extent of ST-T segment deviation. Four of nine patients had chest pain during minocycline injection. None had ST-T segment changes. Minocycline causes a marked, transient injury to the epicardial-pericardial surface. Our animal and in vitro studies indicate that this acute injury is probably partly related to the acidic pH of the minocycline solution. Our experimental findings suggest that this minocycline-induced injury may be reduced by raising the pH of the solution either ex vivo (eg, by mixing minocycline with previously withdrawn pericardial fluid) or in vivo (eg, by leaving 200 to 300 mL of pericardial fluid prior to minocycline injection). Limited experience in the human with malignant cardiac tamponade indicates that intrapericardial minocycline is usually well tolerated, although severe chest pain may appear.