Colorimetric detection of both total genomic and loci-specific DNA methylation from limited DNA inputs

Background: Aberrant DNA methylation marks are potential disease biomarkers, and detecting both total genomic and gene-specific DNA methylation can aid in clinical decisions. While a plethora of methods exist in research, simpler, more convenient alternatives are needed to enhance both routine diagnostics and research. Results: Herein, we describe colorimetric assays using methyl-binding domain (MBD) proteins for rapid and convenient evaluation of total genomic and gene-specific methylation from 50\ua0ng or less DNA input in under 2\ua0h. As little as 5\ua0% methylation differences can be detected and are enhanced by a novel MBD protocol for improved specificity. Our assays could differentiate naïve from de-methylating drug-treated cells and detect the presence of a methylated prostate cancer biomarker in the urine. Finally, the assay was evolved onto disposable screen-printed electrodes for convenient detection of gene-specific methylation in urine. Conclusions: Rapid MBD-based colorimetric and electrochemical approaches to detect DNA methylation from limited samples were successfully demonstrated and applied to clinical samples. We envision that the ease, low sample requirements and speed of these assays could have both clinical and research-wide applications

Modified critical correlations close to modulated and rough surfaces

Correlation functions are sensitive to the presence of a boundary. Surface modulations give rise to modified near surface correlations, which can be measured by scattering probes. To determine these correlations, we develop a perturbative calculation in deformations in height from a flat surface. The results, combined with a renormalization group around four dimensions, are also used to predict critical behavior near a self-affinely rough surface. We find that a large enough roughness exponent can modify surface critical behavior.Comment: 4 pages, 1 figure. Revised version as published in Phys. Rev. Lett. 86, 4596 (2001

Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies

Universality for 2D Wedge Wetting

We study 2D wedge wetting using a continuum interfacial Hamiltonian model which is solved by transfer-matrix methods. For arbitrary binding potentials, we are able to exactly calculate the wedge free-energy and interface height distribution function and, thus, can completely classify all types of critical behaviour. We show that critical filling is characterized by strongly universal fluctuation dominated critical exponents, whilst complete filling is determined by the geometry rather than fluctuation effects. Related phenomena for interface depinning from defect lines in the bulk are also considered.Comment: 4 pages, 1 figur

Geometry dominated fluid adsorption on sculptured substrates

Experimental methods allow the shape and chemical composition of solid surfaces to be controlled at a mesoscopic level. Exposing such structured substrates to a gas close to coexistence with its liquid can produce quite distinct adsorption characteristics compared to that occuring for planar systems, which may well play an important role in developing technologies such as super-repellent surfaces or micro-fluidics. Recent studies have concentrated on adsorption of liquids at rough and heterogeneous substrates and the characterisation of nanoscopic liquid films. However, the fundamental effect of geometry has hardly been addressed. Here we show that varying the shape of the substrate can exert a profound influence on the adsorption isotherms allowing us to smoothly connect wetting and capillary condensation through a number of novel and distinct examples of fluid interfacial phenomena. This opens the possibility of tailoring the adsorption properties of solid substrates by sculpturing their surface shape.Comment: 6 pages, 4 figure

Correlation functions near Modulated and Rough Surfaces

In a system with long-ranged correlations, the behavior of correlation functions is sensitive to the presence of a boundary. We show that surface deformations strongly modify this behavior as compared to a flat surface. The modified near surface correlations can be measured by scattering probes. To determine these correlations, we develop a perturbative calculation in the deformations in height from a flat surface. Detailed results are given for a regularly patterned surface, as well as for a self-affinely rough surface with roughness exponent $\zeta$. By combining this perturbative calculation in height deformations with the field-theoretic renormalization group approach, we also estimate the values of critical exponents governing the behavior of the decay of correlation functions near a self-affinely rough surface. We find that for the interacting theory, a large enough $\zeta$ can lead to novel surface critical behavior. We also provide scaling relations between roughness induced critical exponents for thermodynamic surface quantities.Comment: 31 pages, 2 figure

Electrochemically controlled growth and positioning of suspended collagen membranes

Two independently recognized in vitro polymer aggregation variables, electric field and pH, can be used in concert to produce suspended membranes from solutions of type I collagen monomers, without need of a supporting substrate. A collagen network film can form at the alkalineacidic pH interface created during the normal course of water electrolysis with parallel plate electrodes, and the anchoring location can be controlled by adjusting the bulk electrolyte pH. Electrosynthesized films remain intact upon drying and rehydration and function as ion separation membranes even in submillimeter channels. This approach could benefit lab-on-a-chip technologies for rational placement of membranes in microfluidic devices

New Colloidal Lithographic Nanopatterns Fabricated by Combining Pre-Heating and Reactive Ion Etching

We report a low-cost and simple method for fabrication of nonspherical colloidal lithographic nanopatterns with a long-range order by preheating and oxygen reactive ion etching of monolayer and double-layer polystyrene spheres. This strategy allows excellent control of size and morphology of the colloidal particles and expands the applications of the colloidal patterns as templates for preparing ordered functional nanostructure arrays. For the first time, various unique nanostructures with long-range order, including network structures with tunable neck length and width, hexagonal-shaped, and rectangular-shaped arrays as well as size tunable nanohole arrays, were fabricated by this route. Promising potentials of such unique periodic nanostructures in various fields, such as photonic crystals, catalysts, templates for deposition, and masks for etching, are naturally expected

A microfluidic-SERSplatform for isolation and immuno-phenotyping of antigen specific T-cells

T-cells play a major role in host defense mechanisms against many diseases. With the current growth of immunotherapy approaches, there is a strong need for advanced technologies to detect and characterize these immune cells. Herein, we present a simple approach for the isolation of antigen specific T-cells from the complex biological sample based on T-cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interaction. Subsequently, we characterize those antigen specific T-cells by profiling TCR expression heterogeneity. Our approach utilizes an alternating current electrohydrodynamic (ac-EHD) based microfluidic platform for isolation and surface enhanced Raman scattering (SERS) for TCR expression profiling. The use of ac-EHD enables specific isolation of T-cells by generating a nanoscopic shear force at the double layer of the sensing surface which enhances the frequency of pMHC and TCR interactions and consequently shears off the nonspecific targets. TCR expression profiling of the isolated T-cells was performed by encoding them with SERS-labelled pMHCs followed by SERS detection in bulk as well as in single T-Cell. In proof-of-concept experiments, 56.93 ± 7.31% of the total CD4+T-cells were captured from an excess amount of nonspecific cells (e.g., PBMCs) with high specificity and sensitivity (0.005%). Moreover, TCR analysis data using SERS shows the heterogeneity in the T-cell receptor expression which can inform on the activation status of T-cells and the patient’s response to immunotherapy. We believe that this approach may hold potential for numerous applications towards monitoring immune status, understanding therapeutic responses,and effective vaccine development