A Journey with LGMD: From Protein Abnormalities to Patient Impact.

The limb-girdle muscular dystrophies (LGMD) are a collection of genetic diseases united in their phenotypical expression of pelvic and shoulder area weakness and wasting. More than 30 subtypes have been identified, five dominant and 26 recessive. The increase in the characterization of new genotypes in the family of LGMDs further adds to the heterogeneity of the disease. Meanwhile, better understanding of the phenotype led to the reconsideration of the disease definition, which resulted in eight old subtypes to be no longer recognized officially as LGMD and five new diseases to be added to the LGMD family. The unique variabilities of LGMD stem from genetic mutations, which then lead to protein and ultimately muscle dysfunction. Herein, we review the LGMD pathway, starting with the genetic mutations that encode proteins involved in muscle maintenance and repair, and including the genotype-phenotype relationship of the disease, the epidemiology, disease progression, burden of illness, and emerging treatments.

Homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes.

Herein we report the development of a simple, rapid, homogeneous, and sensitive detection system for DNA based on the scattering properties of silver-amplified gold nanoparticle probes. The assay uses DNA-functionalized magnetic particle probes that act as scavengers for target DNA, which can be collected via a magnetic field. Once the DNA targets are isolated from the initial sample, they are sandwiched via hybridization by a second set of probes. The latter probes are 13-nm gold nanoparticles modified with a different target complementary DNA. Excess probes are removed through repetitive washing steps. The gold particles are dispersed in solution by dehybridization, corresponding to an assumed 1:1 ratio with the target DNA. Electroless deposition of silver on the surface of the gold probes results in particle growth, which increases their scattering efficiency with time. The scattering efficiency and the extinction signatures of the particle sizes are monitored as a function of time and correlated with target concentration. The limit of detection for this novel assay was determined to be 10 fM.

A bio-barcode assay for on-chip attomolar-sensitivity protein detection.

Functionalized nanoparticles hold great promise in realizing highly sensitive and selective biodetection. We report a single disposable chip which is capable of carrying out a multi-step process that employs nanoparticles--a bio-barcode assay (BCA) for single protein marker detection. To illustrate the capability of the system, we tested for the presence of prostate specific antigen (PSA) in buffer solution and goat serum. Detection was accomplished at PSA concentrations as low as 500 aM. This corresponds to only 300 copies of protein analytes using 1 microL total sample volume. We established that the on-chip BCA for PSA detection offers four orders of magnitude higher sensitivity compared to commercially available ELISA-based PSA tests.

Gold nanoparticle probes for the detection of nucleic acid targets.

BACKGROUND: Advances in nanoscience are having a significant impact on many scientific fields and are resulting in the development of a variety of important technologies. This impact is particularly large in the field of biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection, with DNA- or protein-functionalized gold nanoparticles used as the target-specific probes. METHODS: Assays provide an analysis of the unique biophysical properties displayed by gold nanoparticles and have advantages over conventional detection methods (e.g., molecular fluorophores, real-time polymerase chain reaction, RT-PCR, enzyme linked immunosorbent assays, ELISAs, gel electrophoresis, and microarray technologies). CONCLUSION: Some of the advantages include the assays' PCR-like sensitivity, their selectivity for target sequences, their capacity for massive multiplexing, their time efficiency, and most importantly, their ability to be performed at the point of care.

A bio-bar-code assay based upon dithiothreitol-induced oligonucleotide release.

The recently developed bio-bar-code assay for the PCR-less detection of protein and nucleic acid targets has been shown to be extraordinarily sensitive, exhibiting low attomolar sensitivity for protein targets and high zeptomolar sensitivity for nucleic acid targets. In the case of DNA detection, the original assay relies on three distinct oligonucleotide strands on a single nanoparticle for target identification and signal amplification. Herein, we report the development of a new nanoparticle probe that can be used in the bio-bar-code assay, which requires only one thiolated oligonucleotide strand. This new assay relies on the ability to liberate the adsorbed thiolated oligonucleotides from the gold nanoparticle surface with dithiothreitol (DTT), which simplifies the assay and increases its quantitative capabilities. The utility of this new DTT-based system is demonstrated by detecting a mock mRNA target using both fluorescent and scanometric assay readouts. When the scanometric readout is used, the sensitivity of the assay is 7 aM and quantification can be accomplished over the low-attomolar to the mid-femtomolar concentration range.

Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease.

The recently developed ultrasensitive bio-barcode assay was used to measure the concentration of amyloid-beta-derived diffusible ligands (ADDLs), a potential soluble pathogenic Alzheimer's disease (AD) marker, in the cerebrospinal fluid (CSF) of 30 individuals. ADDL concentrations for the subjects diagnosed with AD were consistently higher than the levels in the CSF taken from nondemented age-matched controls. Studies of ADDLs or for any other potential pathogenic AD markers in CSF have not been possible because of their low concentration in CSF (<1 pM). This study is a step toward a diagnostic tool, based on soluble pathogenic markers for the debilitating disease.

Electron hopping dynamics in monolayer-protected au cluster network polymer films by rotated disk electrode voltammetry.

Electrons are transported within polymeric films of alkanethiolate monolayer-protected Au clusters (MPCs) by electron hopping (self-exchange) between the metal cores. The surrounding monolayers, the molecular linkers that generate the network polymer film, or both, presumably serve as tunneling bridges in the electron transfers. This paper introduces a steady-state electrochemical method for measuring electron hopping rates in solvent-wetted and swollen, ionically conductive MPC films. The films are network polymer films of nanoparticles, coated on a rotated disk electrode that is contacted by a solution of a redox species (decamethylferrocene, CpFe). Controlling the electrode potential such that the film mediates oxidation of the redox probe can force control of the overall current onto the rate of electron hopping within the film, which is characterized as the apparent electron diffusion coefficient D(E). D(E) is translated into an apparent electron hopping rate k(ET) by a cubic lattice model. The experiment is applied to MPC network polymer films linked by alpha,omega-alkanedithiolates and by metal ion-carboxylate connections. We evaluate the dependencies of apparent hopping rate on CpFe concentration, film thickness, electrode potential relative to the CpFe formal potential, film-swelling solvent, and temperature. The apparent hopping rates are in the 10(4)-10(5) s(-)(1) range, which is slower than those for the same kind of MPC films, but in a dry (nonswollen) state measured by electronic conductivities.

Hexanethiolate monolayer protected 38 gold atom cluster.

The nucleation-growth-passivation Brust reaction has been modified so as to enrich the product in useful quantities of a 38-atom gold nanoparticle coated with a hexanethiolate monolayer. Two modifications are described, using -78 degrees C reduction temperature and a hyperexcess of thiol. Compositional evidence is presented that establishes the product as a Au38(C6)24 hexanethiolate monolayer protected cluster (MPC), based on transmission electron microscopy, laser ionization-desorption mass spectrometry, thermogravimetric analysis, and elemental analysis. Reverse phase HPLC confirms the relatively good monodispersity of the MPC products, but high-resolution double-column HPLC reveals that the MPCs are a mixture of closely related but chromatographically distinct products. Voltammetry, low energy spectrophotometry, and spectroelectrochemistry reveal, respectively, a 1.6 eV electrochemical energy gap between the first oxidation and the first reduction, an optical HOMO-LUMO energy absorbance edge at 1.3 eV, and a bleaching of optical absorbance near the 1.3 eV band edge that accompanies electrochemical oxidation of the nanoparticle.