Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.

BACKGROUND: Decreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens. METHODS: GALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone. GALC activity was demonstrated on newborn DBS specimens, known Krabbe disease patient specimens, proficiency testing and quality control samples. RESULTS: We present data on characterization of the novel substrate and assay, including pH optimization and enzyme kinetics using a fluorimetric profile. Single and multi-day precision analyses revealed tight analytical measurements with %CV ranging from 5.2% to 14.1%. GALC enzyme activity was linear over the range of 0.31 - 12.04 µmol/l/h with a limit of detection of 0.066 µmol/l/h. Our results with this assay show a clear discrimination between GALC activities in samples from Krabbe disease patients versus presumed normal newborn samples. CONCLUSIONS: A fluorimetric assay for GALC enzyme activity measurement on dried blood spot specimens is feasible. Improvements to the assay including novel substrate design, increased substrate concentration and removal of sodium chloride maximize the specificity of the assay and minimize interference from ß-galactosidase.

Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns.

BACKGROUND: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods. METHODS: We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. RESULTS: Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. CONCLUSIONS: A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

Multiplexed SNP genotyping using nanobarcode particle technology.

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.

Use of nanobarcodes particles in bioassays.

We have developed striped metal nanoparticles, Nanobarcodes particles, which can act as encoded substrates in multiplexed assays. These particles are metallic, encodeable, machine-readable, durable, submicron-sized tags. The power of this technology is that the particles are intrinsically encoded by virtue of the difference in reflectivity of adjacent metal stripes. This chapter describes protocols for the attachment of biological molecules, and the subsequent use of the Nanobarcodes particles in bioassays.

Encoded metal nanoparticle-based molecular beacons for multiplexed detection of DNA.

In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3' universal fluorescence dye. A 5' thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3' fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.

Particles for multiplexed analysis in solution: detection and identification of striped metallic particles using optical microscopy.

In this report, we present data demonstrating that cylindrical metallic particles, with various submicrometer striping patterns, may be readily distinguished in an optical microscope. Accurate particle identification is discussed relative to synthesis reproducibility and the limitations of optical microscopes. Results from a library of these particles, of which over 100 different striping patterns have been produced, are presented. For these particles, made with Au and Ag stripes, more than 70 patterns may be identified with greater than 90% accuracy. The ability to chemically modify the surface of these particles, making them useful for bioanalytical measurements, is also demonstrated. Finally, we discuss improvements in our manufacturing and identification processes that will lead to both larger numbers of striping patterns and improved identification accuracy.

New algorithms for processing and peak detection in liquid chromatography/mass spectrometry data.

Two new algorithms for automated processing of liquid chromatography/mass spectrometry (LC/MS) data are presented. These algorithms were developed from an analysis of the noise and artifact distribution in such data. The noise distribution was analyzed by preparing histograms of the signal intensity in LC/MS data. These histograms are well fit by a sum of two normal distributions in the log scale. One new algorithm, median filtering, provides increased performance compared to averaging adjacent scans in removing noise that is not normally distributed in the linear scale. Another new algorithm, vectorized peak detection, provides increased robustness with respect to variation in the noise and artifact distribution compared to methods based on determining an intensity threshold for the entire dataset. Vectorized peak detection also permits the incorporation of existing algorithms for peak detection in ion chromatograms and/or mass spectra. The application of these methods to LC/MS spectra of complex biological samples is described.