Technology

Extrinsic Raman label (ERL) reagents combined with the Nanoraman instrument provide a complete Ramanprobes system capable of detecting specific components of complex biomolecular assemblies with exquisite sensitivity and selectivity.  Ramanprobes systems offer significant advantages over current state-of-the-art fluorescent systems in sensitivity, analytical concurrency, instrumentation simplification, and development flexibility.

ERL labeled antibodies are important for both research and diagnostic applications because they are amenable to sensitive detection techniques.  Though the current ERL reagent line focuses only on antibody labeling, future products will include the labeling of antigens, nucleic acids, lectins, and aptamers to name a few.  Considered a primary detection reagent, our technique uses the principle of a tight association labeling to confer existence of the antigen.

ERL reagents are based on our novel strategy exploiting the strong surface enhanced Raman scattering (SERS)-derived signal from organic dyes (i.e., reporter molecules) that are immobilized on Au nanoparticles and subsequently coupled to the appropriate biospecific species.  The identity of each analyte is determined from the characteristic SERS spectrum of the nanoparticle-bound reporter species linked to the tracer antibody, with each antigen then quantified by the spectral intensity of reporter species.  The advantages of this strategy largely reflect two unique features of SERS.  First, the widths of Raman spectral bands for nearly all organic compounds are typically 10-100 times narrower than those of fluorescence, a characteristic that minimizes the potential for spectral overlap in the response from the different labels.  Second, the intensities of the SERS response for immobilized reporters can surpass those of fluorescence dyes. 

Our Advantages Over Existing Fluorescence Techniques

We are a direct replacement to fluorescence labeling technology.  The following features of the Ramanprobes system improves current fluorescence labeling technology by:

	The optimum excitation wavelength for SERS depends on nanoparticle composition and size, but not the immobilized dye identity, which therefore requires only one excitation source (e.g., low cost diode laser) for all assays.
	Raman scattering is not affected by fluorescence quenchers (e.g., oxygen) and is less prone to photobleaching, which not only facilitates the application of SERS to a wide range of sample matrices but also enables signal averaging for extended time periods to lower limits of detection.
	SERS with Au substrates requires long wavelength excitation (i.e., red to near infrared region) for coupling with the surface plasmon of the substrate, which reduces background from native fluorescence from the sample.
	Ramanprobes systems have been shown to be more sensitive.  Current levels of detection are down to 1 molecule in 3 seconds.

Typical Operation

The user combines monoclonal or polyclonal antibodies for the target analyte with the ERL 1336 reagent creating a labeled antibody.  The Au Analysis Plate is also prepared with the target monoclonal or polyclonal antibodies.  The analyte (target) solution is added to the plate.  After a predetermined amount of time, the ERL 1336 reagent prepared in step 1 is then added, and then the plate is washed.  The plate is then placed under the Raman microscope for analysis.  The instrument utilizes a highly monochromatic laser to illuminate the sample and collect the Raman scattered light.  Because the ERL 1336 label is surface enhanced, its signal will be approximately 10¹⁴ times stronger than any other Raman signal collected.  Because the Raman spectra of the ERL labels are very narrow, multiple reagents with different Raman responses (peaks) may be used with different antibodies (but illuminated concurrently by the same Raman system). 

Publications

Immunoassay Readout Method Using Extrinsic Raman Labels Adsorbed on Immunogold Colloids, Jing Ni, Robert J. Lipert, G. Brent Dawson, and Marc D. Porter,  Anal. Chem. 1999, 71, 4903-4908.

Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection, Yunwei Charles Cao, Rongchao Jin, Chad A. Mirkin, Science 2002, 297, 1536-1540.

Femtomolar Detection of Prostate Specific Antigen: an Immunoassay Based on Surface-Enhanced Raman Scattering and Immunogold Labels, Desiree Grubisha, Robert J. Lipert, Hye-Young Park, Jeremy Driskell, and Marc D. Porter, Anal. Chem. 2003, 75(21), 5936-5943.

Single Particle Raman Measurements of Gold Nanoparticles Used in Surface-Enhanced Raman Scattering (SERS)-Based Sandwich Immunoassays, Hye-Young Park, Robert J. Lipert, and Marc D. Porter, SPIE Nanosensing: Materials and Devices 2005, 5593, 464-477.

Low Level Detection of Viral Pathogens by a Surface-Enhanced Raman Scattering Based Immunoassay, J. D. Driskell, K. M. Kwarta, R. J. Lipert, M. D. Porter, J. Neill, and J. Ridpath, Anal. Chem. 19 6147-54 (2005).

Ultrasensitive Immunoassays Based on Surface-Enhanced Raman Scattering by Immunogold Labels, Park, HY; Driskell, JD; Kwarta, KM; Lipert, RJ; Porter, MD; Schoen, C; Neill, JD; Ridpath, JF, Topics in Applied Physics, 2006, vol. 103, pp. 427-446.

Detection of Viruses: Atomic Force Microscopy and Surface Enhanced Raman Spectroscopy, Porter, MD; Driskell, JD; Kwarta, KM; Lipert, RJ; Neill, JD; Ridpath, JF, Developments in Biologicals, 2006, vol. 126, pp. 31-40.

Labeled Gold Nanoparticles Immobilized at Smooth Metallic Substrates: Systematic Investigation of Surface Plasmon Resonance and Surface-Enhanced Raman Scattering, Driskell, JD; Lipert, RJ; Porter, MD, Journal of Physical Chemistry B, 2006, vol. 110, no. 35, pp. 17444-17451.