Competitive surface-enhanced Raman scattering assay for the 1,25-dihydroxy metabolite of vitamin D3.

This paper describes the development and preliminary testing of a competitive surface-enhanced Raman scattering (SERS) immunoassay for calcitriol, the 1,25-dihydroxy metabolite (1,25-(OH)(2)-D(3)) of vitamin D(3). Deficiencies in 1,25-(OH)(2)-D have been linked to renal disease, while elevations are linked to hypercalcemia. Thus, there has been a sharp increase in the clinical demand for measurements of this metabolite. The work herein extends the many attributes of SERS-based sandwich immunoassays that have been exploited extensively in the detection of large biolytes (e.g., DNA, proteins, viruses, and microorganisms) into a competitive immunoassay for the low level determination of a small biolyte, 1,25-(OH)(2)-D(3) (M(w) = 416 g mol(-1)). The assay uses surface modified gold nanoparticles as SERS labels, and has a dynamic range of 10-200 pg mL(-1) and a limit of detection of 8.4 ± 1.8 pg mL(-1). These analytical performance metrics match those of tests for 1,25-(OH)(2)-D(3) that rely on radio- or enzyme-labels, while using a much smaller sample volume and eliminating the disposal of radioactive wastes. Moreover, the SERS-based data from pooled-patient sera show strong agreement with that from radioimmunoassays. The merits and potential utility of this new assay are briefly discussed.

Increased sensitivity of bacterial detection in cerebrospinal fluid by fluorescent staining on low-fluorescence membrane filters.

A membrane-filter-based, fluorescent Gram stain method for bacterial detection in cerebrospinal fluid samples was developed and evaluated as a rapid, sensitive alternative to standard Gram stain protocols. A recently developed, modified version of the aluminium oxide membrane Anopore with low-fluorescence optical properties showed superior performance in this application. Other aspects of the fluorescent Gram stain system that were evaluated include membrane filter selection, strategies to reduce fluorescence fading and the effect of patient blood cells on bacterial detection in the fluorescently stained cerebrospinal fluid samples. The combination of the membrane filter's bacteria-concentrating ability and absolute retention along with high-contrast, fluorescent Gram discriminating dyes enabled rapid bacterial detection and Gram discrimination, with a 1-1.5 order of magnitude increase in the bacterial concentration limit of detection.

The 104-well microplate.

The 96-well microplate is a ubiquitous tool in the laboratory; its use is so extensive that in a limited number of situations it can be restrictive. Consider the situation where 96 samples need analysis or a downstream process in which the 96-well format leaves no space for additional standards or controls in the upstream 96-well processing. Consequently, plates are split or sample number reduced thereby incurring additional cost for plates, reagents, standards, controls, sample tracking, data files, and time to analyze the entire plate. A simple solution is proposed with the development of a companion 8 × 13-array microplate. The 104-well microplate was developed within the American National Standards Institute/Society for Biomolecular Science standards as to plate geometry and dimension, including well spacing (9 mm) with the exception that the columns have been shifted 4.5mm to the left to accommodate the 13th column. The extra column allows for additional standards/controls without modifying chemistry, incorporating additional plates or changing to a 384-well plate. We show negligible difference (-0.0003 optical density) when comparing mean absorbance readings in 96- and 104-well format. We demonstrate use of the 104-well plate in a 96-well environment by incorporating it in an enzyme-linked immunosorbent assay on a standard liquid handler. Results from the assay show no difference between formats (y=1.039x-0.004, r=0.997). Although the 104 plate was not created to supplant the 96-well standard, we conclude that the 104 plate can be incorporated into the 96-well environment without significant change in existing systems.

Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping.

BACKGROUND: Additional instruments have become available since instruments for DNA melting analysis of PCR products for genotyping and mutation scanning were compared. We assessed the performance of these new instruments for genotyping and scanning for mutations. METHODS: A 110-bp fragment of the beta-globin gene including the sickle cell anemia locus (HBB c. 20A>T) was amplified by PCR in the presence of LCGreen Plus or SYBR Green I. Amplicons of 4 different genotypes [wild-type, homozygous, and heterozygous HBB c. 20A>T and double-heterozygote HBB c. (9C>T; 20A>T)] were melted on 7 different instruments [Applied Biosystems 7300, Corbett Life Sciences Rotor-Gene 6500HRM, Eppendorf Mastercycler RealPlex4S, Idaho Technology LightScanner (384 well), Roche LightCycler 480 (96 and 384 well) and Stratagene Mx3005p] at a rate of 0.61 degrees C/s or when this was not possible, at 0.50 degrees C steps. We evaluated the ability of each instrument to genotype by melting temperature (Tm) and to scan for heterozygotes by curve shape. RESULTS: The ability of most instruments to accurately genotype single-base changes by amplicon melting was limited by spatial temperature variation across the plate (SD of Tm = 0.020 to 0.264 degrees C). Other variables such as data density, signal-to-noise ratio, and melting rate also affected heterozygote scanning. CONCLUSIONS: Different instruments vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole amplicon melting analysis. Instruments specifically designed for high-resolution melting, however, displayed the least variation, suggesting better genotyping accuracy and scanning sensitivity and specificity.

Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes.

BACKGROUND: DNA melting analysis for genotyping and mutation scanning of PCR products by use of high-resolution instruments with special "saturation" dyes has recently been reported. The comparative performance of other instruments and dyes has not been evaluated. METHODS: A 110-bp fragment of the beta-globin gene including the sickle cell anemia locus (A17T) was amplified by PCR in the presence of either the saturating DNA dye, LCGreen Plus, or SYBR Green I. Amplicons of 3 different genotypes (wild-type, heterozygous, and homozygous mutants) were melted on 9 different instruments (ABI 7000 and 7900HT, Bio-Rad iCycler, Cepheid SmartCycler, Corbett Rotor-Gene 3000, Idaho Technology HR-1 and LightScanner, and the Roche LightCycler 1.2 and LightCycler 2.0) at a rate of 0.1 degrees C/s or as recommended by the manufacturer. The ability of each instrument/dye combination to genotype by melting temperature (Tm) and to scan for heterozygotes by curve shape was evaluated. RESULTS: Resolution varied greatly among instruments with a 15-fold difference in Tm SD (0.018 to 0.274 degrees C) and a 19-fold (LCGreen Plus) or 33-fold (SYBR Green I) difference in the signal-to-noise ratio. These factors limit the ability of most instruments to accurately genotype single-nucleotide polymorphisms by amplicon melting. Plate instruments (96-well) showed the greatest variance with spatial differences across the plates. Either SYBR Green I or LCGreen Plus could be used for genotyping by T(m), but only LCGreen Plus was useful for heterozygote scanning. However, LCGreen Plus could not be used on instruments with an argon laser because of spectral mismatch. All instruments compatible with LCGreen Plus were able to detect heterozygotes by altered melting curve shape. However, instruments specifically designed for high-resolution melting displayed the least variation, suggesting better scanning sensitivity and specificity. CONCLUSION: Different instruments and dyes vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole-amplicon melting analysis.