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.

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.