Photonic crystals on copolymer film for label-free detection of DNA hybridization.

The presence of a single-nucleotide polymorphism in Apolipoprotein E4 gene is implicated with the increased risk of developing Alzheimer's disease (AD). In this study, detection of AD-related DNA oligonucleotide sequence associated with Apolipoprotein E4 gene sequence was achieved using localized-surface plasmon resonance (LSPR) on 2D-Photonic crystal (2D-PC) and Au-coated 2D-PC surfaces. 2D-PC surfaces were fabricated on a flexible copolymer film using nano-imprint lithography (NIL). The film surface was then coated with a dual-functionalized polymer to react with surface immobilized DNA probe. DNA hybridization was detected by monitoring the optical responses of either a Fresnel decrease in reflectance on 2D-PC surfaces or an increase in LSPR on Au-coated 2D-PC surfaces. The change in response due to DNA hybridization on the modified surfaces was also investigated using mismatched and non-complementary oligonucleotides sequences. The proof-of-concept results are promising towards the development of 2D-PC on copolymer film surfaces as miniaturized and wearable biosensors for various diagnostic and defense applications.

Au nanostructured surfaces for electrochemical and localized surface plasmon resonance-based monitoring of α-synuclein-small molecule interactions.

In this proof-of-concept study, the fabrication of novel Au nanostructured indium tin oxide (Au-ITO) surfaces is described for the development of a dual-detection platform with electrochemical and localized surface plasmon resonance (LSPR)-based biosensing capabilities. Nanosphere lithography (NSL) was applied to fabricate Au-ITO surfaces. Oligomers of α-synuclein (αS) were covalently immobilized to determine the electrochemical and LSPR characteristics of the protein. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed using the redox probe [Fe(CN)6](3-/4-) to detect the binding of Cu(II) ions and (-)-epigallocatechin-3-gallate (EGCG) to αS on the Au-ITO surface. Electrochemical and LSPR data were complemented by Thioflavin-T (ThT) fluorescence, surface plasmon resonance imaging (SPRi), and transmission electron microscopy (TEM) studies. EGCG was shown to induce the formation of amorphous aggregates that decreased the electrochemical signals. However, the binding of EGCG with αS increased the LSPR absorption band with a bathochromic shift of 10-15 nm. The binding of Cu(II) to αS enhanced the DPV peak current intensity. NSL fabricated Au-ITO surfaces provide a promising dual-detection platform to monitor the interaction of small molecules with proteins using electrochemistry and LSPR.

Au nanoparticle-modified DNA sensor based on simultaneous electrochemical impedance spectroscopy and localized surface plasmon resonance.

Electrochemical impedance spectroscopy (EIS) and localized surface plasmon resonance (LSPR) were performed on the same Au nanoparticle (AuNP)-modified indium tin oxide (ITO) coated glass surfaces. Cyclic voltammetry was applied to electrodeposit AuNPs on ITO surface directly. The surface plasmon band characterization of AuNPs was initially studied by controlling the electrodeposition conditions. It was found that the size of AuNP clusters was significantly affected by the applied potential and KCl concentration in solution. The dual-detection platform was applied to detect DNA hybridization related to a specific point mutation in apolipoprotein E gene (ApoE), which was related to the progression of Alzheimer's disease. The preliminary results facilitate the development of a versatile biosensor that can be easily miniaturized and integrated into a high-throughput diagnostic device.

LED-based interferometric reflectance imaging sensor for the detection of amyloid-ß aggregation.

Self-aggregation of amyloid-ß (Aß) plays an important role in the pathogenesis of Alzheimer's disease (AD). Small molecule inhibitors of Aß fibril formation reduce the Aß-mediated neurotocixity. In this report, the interaction of amyloid-ß (Aß) with well-described modulators, (-)epigallocatechin-3-gallate (EGCG) and Zn(ii), was detected using a LED-based interferometric reflectance imaging sensor (LED-IRIS) in a high-throughput and real-time format. Nucleation-based fibril growth strategy was employed, as the "seeds" of Aß were prepared in the presence of EGCG and Zn(ii). The seeds were then covalently immobilized on the chip surface. Using microfluidics, Aß oligomers were exposed onto the seeds resulting in the elongation of fibrils, which was detected as the increase in the spot height. Monitoring the changes on the chip surface enabled to detect the efficacy of modulators to inhibit or facilitate the growth of Aß fibrils. The proof-of-concept study reported here introduces a novel platform to facilitate the screening of small molecules towards the discovery of promising AD therapeutics.

Electroanalysis of the interaction between (-)-epigallocatechin-3-gallate (EGCG) and amyloid-ß in the presence of copper.

The misfolding of amyloid-beta (Aß) peptide is one of the pathological hallmarks of Alzheimer's disease (AD). Polyphenols are strong antioxidants and metal chelators, with characteristics that are of beneficial therapeutic values for their development as candidates targeting neurodegenerative and metal-induced diseases. We have demonstrated here the electrochemical properties of a green tea component, (-)-epigallocatechin-3-gallate (EGCG), and its potent activity on Aß peptides. Characterization of early interactions (≤48 h) between EGCG and Aß was conducted using square wave voltammetry (SWV). The interaction of Cu(ii) ions with the Tyr-10 residue of Aß was shown to be affected by surrounding His residues. Morphological changes due to the binding of EGCG and Cu(II) were also elucidated using transmission electron microscopy (TEM). Electroanalytical techniques are promising for facilitating the investigation of metals and flavonoids in drug screening studies.

Surface plasmon resonance imaging of amyloid-ß aggregation kinetics in the presence of epigallocatechin gallate and metals.

A number of human protein misfolding disorders, including Alzheimer's disease (AD), are closely related to the accumulation of ß-sheet-rich amyloid fibrils or aggregates. Neuronal toxicity in AD has been linked to the interactions of amyloid-ß (Aß) with metals, especially Zn(2+), Cu(2+), and Fe(3+), which leads to the production of reactive oxygen species. Nucleation-dependent Aß aggregation, or "seeding", is thought to propagate fibril formation. In this surface plasmon resonance imaging (SPRi) study, we have shown that the fibril seeds formed with the incubation of Aß in the presence of metals are better at promoting monomer elongation compared to Aß alone or in the presence of a well-described polyphenol, (-)-epigallocatechin-3-gallate (EGCG). This is a novel attempt to simultaneously monitor the effects of multiple modulators on fibril elongation using a single chip. EGCG was shown in transmission electron microscopy (TEM) and thioflavin T (ThT) studies to promote the formation of off-pathway, highly stable unstructured oligomers, supporting the SPRi results. These findings suggest that SPRi provides a promising platform as a screening tool for small molecules that can affect the aggregation pathways in neurodegenerative diseases.

Photonic crystals on copolymer film for bacteria detection.

The development of two-dimensional photonic crystals (PCs) on a copolymer film is described in connection with Fresnel reflection spectroscopy and fluorescence microscopy. Label-free detection of Legionella pneumophila was performed using a PC platform with a detection limit of 200 cells/mL. L. pneumophila is well known as the cause of Legionnaires' disease and a lesser form called Pontiac fever. Since death by L. pneumophila infection depends on the early anti-microbial treatment, rapid diagnosis of this disease is critical for efficient treatment and patient survival. Conventional assays have turn-around times measurable in several hours to days, and are limited in their detection of various serogroups. Due to the recent introduction of regulatory guidelines for routine testing of water cooling towers and treatment facilities, biosensors for the on-field detection of Legionella spp. are highly in demand. The versatile and economical immunochips described here can be easily adapted for the monitoring of L. pneumophila serogroups in clinical and environmental samples in a few minutes.

Oxidative stress effect of dopamine on α-synuclein: electroanalysis of solvent interactions.

The interaction of dopamine (DA) and α-synuclein (α-S) can lead to protein misfolding and neuronal death triggered by oxidative stress relevant to the progression of Parkinson's disease (PD). In this study, interfacial properties associated with DA-induced α-S aggregation under various solution conditions (i.e., pH, ionic strength) were investigated in vitro. The electrochemical oxidation of tyrosine (Tyr) residues in α-S was detected in the presence of DA. DA concentration dependence was analyzed and found to significantly affect α-S aggregation pathways. At low pH, DA was shown to be stable and produced no observable difference in interfacial properties. Between pH 7 and 11, DA promoted α-S aggregation. Significant differences in oxidation current signals in response to high pH and ionic strength suggested the importance of initial interactions in the stabilization of toxic oligomeric structures and subsequent off-pathways of α-S. Our results demonstrate the importance of solution interactions with α-S and the unique information that electrochemical techniques can provide for the investigation of α-S aggregation at early stages, an important step toward the development of future PD therapeutics.

Disposable immunochips for the detection of Legionella pneumophila using electrochemical impedance spectroscopy.

The rapid diagnosis of Legionellosis is crucial for the effective treatment of this disease. Currently, most clinical laboratories utilize rapid immunoassays that are sufficient for the detection of Legionella serogroup 1, but not other clinically relevant serogroups. In this report, the development of a disposable immunochip system is described in connection with electrochemical impedance spectroscopy and fluorescence microscopy. The immunochips were prepared by covalently immobilizing fluorophore-conjugated L. pneumophilaantibodies on Au chips. The analytical performance of the immunochips was optimized as a prescreening tool for L. pneumophila. The versatile immunochips described here can be easily adapted for the monitoring of all Legionella serogroups in clinical and environmental samples.

Optical trapping for the characterization of amyloid-beta aggregation kinetics.

Alzheimer's disease (AD) is marked by the accumulation of neuronal plaques from insoluble amyloid-beta (Aß) peptides. Growing evidence for the role of Aß oligomers in neuronal cell cytotoxicity and pathogenesis has prompted the development of novel techniques to better understand the early stages of aggregation. Near infrared (NIR) optical trapping was applied to characterize the early stages of Aß aggregation in the presence of a ß-sheet intercalating dye, Congo Red (CR), as the fluorescent marker. The integration of fluorescence analysis with NIR optical trapping has provided a new outlook into the first two hours of Aß aggregation.