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. 2016 Jan 7:10:2.
doi: 10.1186/s13036-015-0023-y. eCollection 2016.

Rational design of Raman-labeled nanoparticles for a dual-modality, light scattering immunoassay on a polystyrene substrate

Nathan D Israelsen  1 , Donald Wooley  1 , Cynthia Hanson  1 , Elizabeth Vargis  1
Affiliations

Rational design of Raman-labeled nanoparticles for a dual-modality, light scattering immunoassay on a polystyrene substrate

Nathan D Israelsen et al. J Biol Eng. .

Abstract

Background: Surface-enhanced Raman scattering (SERS) is a powerful light scattering technique that can be used for sensitive immunoassay development and cell labeling. A major obstacle to using SERS is the complexity of fabricating SERS probes since they require nanoscale characterization and optical uniformity. The light scattering response of SERS probes may also be modulated by the substrate used for SERS analysis. A typical SERS substrate such as quartz can be expensive. Polystyrene is a cheaper substrate option but can decrease the SERS response due to interfering Raman emission peaks and high background fluorescence. The goal of this research is to develop an optimized process for fabricating Raman-labeled nanoparticles for a SERS-based immunoassay on a polystyrene substrate.

Results: We have developed a method for fabricating SERS nanoparticle probes for use in a light scattering immunoassay on a polystyrene substrate. The light scattering profile of both spherical gold nanoparticle and gold nanorod SERS probes were characterized using Raman spectroscopy and optical absorbance spectroscopy. The effects of substrate interference and autofluorescence were reduced by selecting a Raman reporter with a strong light scattering response in a spectral region where interfering substrate emission peaks are minimized. Both spherical gold nanoparticles and gold nanorods SERS probes used in the immunoassay were detected at labeling concentrations in the low pM range. This analytical sensitivity falls within the typical dynamic range for direct labeling of cell-surface biomarkers using SERS probes.

Conclusion: SERS nanoparticle probes were fabricated to produce a strong light scattering signal despite substrate interference. The optical extinction and inelastic light scattering of these probes was detected by optical absorbance spectroscopy and Raman spectroscopy, respectively. This immunoassay demonstrates the feasibility of analyzing strongly enhanced Raman signals on polystyrene, which is an inexpensive yet non-ideal Raman substrate. The assay sensitivity, which is in the low pM range, suggests that these SERS probe particles could be used for Raman labeling of cell or tissue samples in a polystyrene tissue culture plate. With continued development, this approach could be used for direct labeling of multiple cell surface biomarkers on strongly interfering substrate platforms.

Keywords: High-throughput; Immunoassay; Multiplexing; Nanoparticle; Raman Spectroscopy; Surface-Enhanced Raman Spectroscopy/Scattering (SERS).

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Figures

Fig. 1
Fig. 1
A comparison of the spectral emission width and structure of fluorescent and SERS probes. The spectral emission width of SERS probe is much narrower than fluorescent probes. The narrow spectral emission width of SERS probes enables the light scattering signal from multiple SERS probes to be detected simultaneously without peak overlap
Fig. 2
Fig. 2
Synthesis schema for the production of SERS probes. The labeling and functionalization of concentrated gold nanoparticles during the fabrication of SERS probes is a multistep process, which can be optimized for use with strongly interfering substrates such as polystyrene
Fig. 3
Fig. 3
Raman reporter selection and polystyrene peak overlap. To avoid substrate interference, Raman reporters were selected that had very little peak overlap with the Raman spectrum of polystyrene. Major peaks observed in the polystyrene spectra are found at 620 cm−1, 1002cm−1, and 1032 cm−1
Fig. 4
Fig. 4
Raman reporter-induced aggregation. When high concentrations of Raman reporter were used during the reporter labeling process, nanoparticle aggregation occurred. The aggregation process results in a LSPR shift and a reduction in LSPR intensity, which can reduce SERS enhancement. DTTC iodide-induced aggregation was observed by monitoring the LSPR peak spectrum of the nanoparticles and by using dynamic light scattering
Fig. 5
Fig. 5
SDS-PAGE analysis of PEGylated antibodies. SDS-PAGE analysis confirmed successful antibody PEGylation. The gel was stained using Coomassie Blue to detect protein (blue/green on the gel) and a barium chloride iodine mixture to detect PEG (brown). The image contrast for Fig. 5 was uniformly adjusted to highlight each individual band in the gel
Fig. 6
Fig. 6
SERS immunoassay development. Fig. 6a presents a visual protocol for the development of a light scattering immunoassay. Step 1: Antigen is non-covalently bound to the polystyrene plate. Step 2: Buffer is used to remove unbound antigen. Step 3: The polystyrene surface is blocked to prevent non-specific binding. Step 4: SERS probe detection antibody is added to the plate. Step 5: Wash buffer is used to remove unbound SERS probe. Step 6: Light scattering from the SERS probe is measured for assay quantification. The resulting assay shows specific binding of the SERS probe to polystyrene embedded human IgG (Fig. 6b)
Fig. 7
Fig. 7
AuNP and AuNR light scattering immunoassay with detection by optical absorbance spectroscopy. After antibody-conjugated gold nanoparticles were bound to the immunoassay plate, the assay response could be detected using optical absorbance spectroscopy. The absorbance spectrum of each well was recorded and the intensity of the major LSPR peaks for both AuNP and AuNR SERS probes was determined (Fig. 7a, c). The LSPR response was correlated to the nanoparticle concentration and a standard curve and sensitivity plot was developed (Fig. 7b, 7d). The curve was fit using five-parameter logistic equation. LLOD values for AuNP and AuNR assays detected using optical absorbance spectroscopy were 22.8 pM and 60.3 pM, respectively
Fig. 8
Fig. 8
AuNP and AuNR light scattering immunoassay with detection by SERS. Using the 785nm custom Raman microscope, the Raman spectra of the AuNP and AuNR SERS probes bound to the polystyrene plate were recorded. Fig. 8a shows the Raman spectrum of DTTC iodide labeled AuNP SERS probes. The spectrum shows characteristic peaks corresponding to DTTC iodide at 493 cm−1 and 508 cm−1 and to polystyrene at 1002 cm−1 and 1032 cm−1. By solving for the area under each reporter peak, an AuNP SERS probe standard curve was developed (Fig. 8b). The curve was fit using a five-parameter logistic equation and the LLOD was calculated as 76.1 pM. The Raman spectra of AuNR SERS probes were also acquired (Fig. 8c). A standard curve and sensitivity plot shows that the LLOD for the AuNR SERS probe assay was 44.7 pM (Fig. 8d)
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