Detection tags based upon surface enhanced Raman scattering provide an alternative to the widely used fluorescence methods. Several aspects of these tags are presented in this report. The tags can be made to display many different spectra, thus they can be used for multiplexed detection schemes. They generate a large enough number of photons to be readily detected, and spectra acquired from mixtures of tags can be analyzed giving accurate amounts of the components. The surface of the tags can be easily modified to present common biological molecules (streptavidin and analogues). Finally, we demonstrate their use to quantitatively detect interleukin-4 (IL4) and interleukin-7 (IL7) in a microarray format.
At Nanoplex Technologies, Inc. we have developed Nanobarcodes particles, which are encodeable, machine-readable, durable, sub-micron sized taggants which have application for document and product security. We will present results on the use of Nanobarcodes particles in a number of authentication and anti-counterfeiting applications. We also focus on the software component in recognition of particles imaged against varied backgrounds.
Modern chemical synthesis and screening technologies have the ability to create large numbers of lead components but still do not answer questions of efficacy, dosing, toxicity and optimal patient population. SurroMed was founded to develop discovery technologies for new biological markers that will answer these questions. Biological markers will be derived from the results of many different assays; cell surface, serum factors and others, many performed using whole blood and other fluids and tissues. We report on the design of a Microvolume Laser Scanning Cytometer (MLSC) and disposable capillary arrays to be used in biological marker discovery. The MLSC machines are used primarily for cell surface assays, though they are suitable for other fluorescence assays as well. Each capillary requires a very small sample volume per assay, less than twenty micro- liters, and so allows hundreds of assays to be performed on a single ten milliliter blood draw. The new MLSC is capable of optimally detecting four fluorescence colors at different scan rates. HeNe excitation and red emission permits the use of whole blood, so that no lysing or cell separation is required. The MLSC instrument and disposable capillary arrays are in routine use for biological marker discovery at SurroMed.
We have been exploring the use of light scattering as a means to detect the binding of nucleic acids to high density DNA probe arrays. Initial work has concentrated on the use of 100 nanometer gold particles conjugated to monoclonal antibodies. A probe array scanner that utilizes an arc lamp source and a `photocopier grade' linear CCD detector has been developed. The optical configuration of the scanner maximizes dynamic range and minimizes optical backgrounds. Initial development of light scattering detection for the p53 cancer gene application shows that functional performance may be obtained that is essentially equivalent to existing fluorescence detection methodology.
We have been exploiting high density oligonucleotide arrays to carry out sequence analysis of genetic material from diverse sources. The method utilizes the hybridization of fluorophore labelled nucleic acids to the array and interpretation of the resulting spatial pattern of fluorescence. Our ability to obtain sequence information from the array is governed by the interplay of the synthesis and hybridization chemistry, the photophysics of the fluorophores and background interferences, and the performance of the fluorescence imaging system. The high photolithographic resolution and large usable area of the synthesis process and the presence of submonolayer coverages of fluorophores dictate that the fluorescence detection system meet several potentially conflicting performance criteria. High spatial resolution, high sensitivity, large field of view, low chromaticity and image distortion, and high dynamic range are required simultaneously. Suitable nucleic acid-fluorophore conjugates should have high absorption cross sections and emission quantum yields, low photobleaching quantum yields, and resistance to transient saturation under intense illumination. Our approaches to the design and photophysical characterization of the detection process will be discussed within the context of improving the volume of sequence information and detection limits.