Detection of DNA targets hybridized to solid surfaces using optical images of liquid crystals.

In this paper, we report a method of detecting DNA targets hybridized to a solid surface by using liquid crystals (LC). The detection principle is based on different interference colors of LC supported on surfaces decorated with single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). However, the contrast between the ssDNA and dsDNA is not obvious, unless DNA-streptavidin complexes are introduced to the dsDNA to increase the surface mass density. Two different approaches of introducing streptavidin to the system are studied and compared. We find that by premixing the biotin-labeled DNA targets with streptavidin prior to the DNA hybridization, branched-streptavidin complexes are formed and clear LC signal can be observed. This LC-based DNA detection principle represents an important step toward the development of a simple, instrument- and fluorophore-free DNA detection method.

Detecting DNA targets through the formation of DNA/CTAB complex and its interactions with liquid crystals.

In this paper, we report the formation of a DNA/cetyl trimethylammonium bromide (CTAB) complex on a solid surface and its interaction with a thin layer of liquid crystals (LC) supported on the surface. Our results show that when the surface is decorated with DNA only, the LC gives a bright image, but when the surface is decorated with the DNA/CTAB complex, the LC becomes dark when the surface density of CTAB is above 5.25 ± 0.13 × 10(13)/cm(2). To exploit this phenomenon for detecting DNA targets, we used a surface decorated with electroneutral PNA probes for capturing DNA targets, and then treated the surface with 0.1 mM of CTAB. In the presence of DNA targets, a PNA/DNA/CTAB complex is formed and that leads to a dark image on the thin layer of the LC supported on the surface. Moreover, DNA targets with a complementary, 1-base mismatch and non-complementary sequence can be differentiated by using this method. This study provides a new principle for the label-free detection of DNA targets without any fluorescent labels.

Enhancing the fluorescence intensity of DNA microarrays by using cationic surfactants.

DNA microarrays have been used as powerful tools in genomics studies and single nucleotide polymorphisms analysis. However, the fluorescence detection used in most conventional DNA microarrays is still limited by its sensitivity. The aim of this study is to use a cationic surfactant, cetyl trimethylammonium bromide (CTAB), to enhance the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA probes immobilized on a DNA microarray. We show that in the presence of CTAB the immobilized FAM-labeled DNA probes is 11-fold brighter than that without exposure to CTAB. Similarly, when we hybridize FAM-labeled DNA targets to a DNA microarray and treat the surface with CTAB solution, the fluorescence intensity shows a 26-fold increase for perfect-match DNA targets. More importantly, the contrast between perfect-match and 1-mismatch DNA is also increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the detection limit of DNA microarrays.

Liquid crystal multiplexed protease assays reporting enzymatic activities as optical bar charts.

We describe a highly sensitive, substrate-specific, label-free, and multiplexed protease assay which reports proteases activities as an optical bar chart, allowing test results to be easily assessed by laymen with the naked eye. First, an oligopeptide microarray having six rows of immobilized oligopeptides, with well-controlled orientations and concentration gradients, is immersed in a buffer solution containing proteases. Then, a thin layer liquid crystal is supported on the microarray to transduce the oligopeptide cleavage event into an optical bar chart of different colors and lengths. This type of optical bar chart provides very rich information such as protease concentration, incubation time, surface densities of oligopeptides, etc. Both trypsin and chymotrypsin can be detected by using this assay within 3 h. The capability of the multiplexed protease assay opens up possibilities for detecting toxins such as botulinum neurotoxins which are known to cleave proteins and affect the docking and fusing synaptic vesicles.

Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption.

In this article, surface coatings derived from homo-bifunctional tri(ethylene glycol) (EG(3)) and hexa(ethylene glycol) (EG(6)) molecules which have two terminal aldehyde groups are reported. These homo-bifunctional molecules can be used to functionalize amine-terminated surfaces through crosslinking one aldehyde group to surface amine groups, while leaving the other aldehyde group available for covalent immobilization of proteins. Best of all, after reducing remaining aldehyde groups on the surface with a reducing agent, sodium borohydride, the surface becomes oligo(ethylene glycol) (OEG)-terminated. The OEG-terminated surface can resist nonspecific protein adsorption, a feature that is often required for many biosensors and biomedical devices. Although some mixed self-assembled monolayers formed from two different organothiols also permit covalent protein immobilization and resist nonspecific protein adsorption, the procedure reported herein requires only one type of homo-bifunctional molecule and can be applied to both silicon and gold surfaces.

A liquid crystal-based sensor for real-time and label-free identification of phospholipase-like toxins and their inhibitors.

We report a liquid crystal (LC)-based sensor for real-time and label-free identification of phospholipase-like toxins. Beta-bungarotoxin exhibits Ca(2+)-dependent phospholipase A(2) activity whereas alpha-bungarotoxin and myotoxin II do not exhibit any phospholipase activity. The sensor can selectively identify beta-bungarotoxin, when it hydrolyzes a phospholipid monolayer self-assembled at aqueous-LC interface, through orientational responses of LCs. As a result, optical signals that reflect the spatial and temporal distribution of phospholipids during the hydrolysis can therefore be generated in a real-time manner. The sensor is very sensitive and requires less than 5pg of beta-bungarotoxin for the detection. When phospholipase A(2) inhibitors are introduced together with beta-bungarotoxin, no orientational response of LCs can be observed. In addition, the regeneration of the sensor can be done without affecting the sensing performance. This work demonstrates a simple and cost-effective LC-based sensor for identifying phospholipase-like toxins and for screening compound libraries to find potential toxin inhibitors.

Optical imaging of surface-immobilized oligonucleotide probes on DNA microarrays using liquid crystals.

In this paper, we report a new label-free method for the imaging of immobilized oligonucleotide probes on DNA microarrays. The imaging principle is based on the disruption of orientations of nematic liquid crystals (LCs), 4-cyano-4'-pentylbiphenyl (5CB), by the immobilized oligonucleotides on a surface. Because LCs are birefringent materials, disruption of their orientations by the immobilized oligonucleotides can manifest as optical signals visible to the naked eye. LC cells with two homeotropic boundary conditions, which align 5CB perpendicularly to both surfaces, were developed to deliver a distinct contrast between a dark background and a bright image caused by the immobilized oligonucleotides. This design also allows the quantification of immobilized oligonucleotide concentrations through the interference colors of LCs. The LC-based imaging method has a good signal-to-noise ratio and a clear distinction between positive and negative results and is nondestructive to the immobilized oligonucleotides. These advantages make it a promising means of assessing the quality of DNA microarrays.