Multifunctional, ultrasensitive plasmonic nanoprobes

Circulating and disseminated biomarkers have surfaced as attractive candidates in cancer diagnosis and prognosis due to the intrinsic advantages of a non-invasive, repeatable liquid biopsy procedures. An exciting new direction in the LBRC is the developmemnt of ultrasensitive plasmonic nanoprobes (PNPs) to aid the detection of circulating biomarkers. Our competitive advantage lies in the overlap between our expertise in high-sensitivity spectroscopic assay development and PNP synthesis. We specifically aim to engineer PNPs with unique Raman scattering and surface plasmon resonance (SPR) properties, allowing quantification of gene-specific methylation markers in cell-free DNA and cell surface-bound antigens, such as CA125, at levels below those achievable today. we strive for features including minimal sample processing requirements, multiplexing capability, and unprecedented sensitivity that will enable rapid quantification of low concentrations of biomarkers in high-throughput settings. Between these SPR-based probes and surface-enhanced Raman scattering (SERS) probes, we aim to develop versatile NP-based blood tests that can be readily translated to clinical laboratory settings for the monitoring of tumor progression during therapy and for the early detection of cancer recurrence.

Multiplexing SERS assay using encoded gold nanostar (GNS) SERS probes

Figure 1: (A) Schematic structure of a SERS tag where a number of Raman reporter molecules, 4-nitrothiophenol (4-NTP), are sandwiched between GNS and thin silica layer. (B) Extinction spectra of bare GNS and SERS tags in aqueous solution, showing the ca. 14 nm red-shift in LSPR absorption maximum of SERS tags (748 nm) compared with the bare GNS (734 nm). (C) Representative TEM image of SERS tags. (D) Raman and SERS spectra of 4-NTP, SERS tag and CA15-3 mAb-modified SERS tag (SERS probe). (E) Schematic illustration of SERS assay for multiplex detection of biomarkers. Imaging is performed over a wide field of the wells in the SERS panel and spatial average of the SERS response is correlated to the levels of corresponding biomarkers. For each measurement, the control is used as the internal standard to calibrate the SERS response. Adapted from [3].

An outstanding challenge in biomedical science is to devise a palette of molecular probes for simultaneous and quantitative detection of an array of molecular species at ultralow, even single-molecule, concentrations. Low-cost biosensors, tailored for rapid, real-time identification of biomarkers, are required in multiple healthcare settings, including routine point-of-care (POC) clinical evaluation, real-time diagnosis of diseases in developing countries, and fast genetic and epigenetic mapping for personalized care. LBRC is committed to exploiting the ability of plasmonic nanoparticles (PNPs) to amplify molecular contrast signals through the combination of large numbers of reporting elements, unique physicochemical properties, the ease of functionalization through facile surface chemistry. PNPs feature strong light scattering many orders of magnitude greater than fluorescent moieties, with plasmonic resonance capable of enhancing the emission and scattering signals of molecules adsorbed on those nanostructures for sensitive and specific detection [1-2]. With breast and ovarian cancers as the driving principal paradigms, LBRC is poised to translate these probes to clinical practice through research in nanomaterials to synthesize bright, and photostable probes; in optics and spectroscopy, to create rapid and portable systems to facilitate POC and in situ molecular detection; and in chemometric analysis, to allow reliable extraction of the signal of interest.

Figure 2: (A) Time-lapse duo-color confocal images of the HeLa cells after co-incubation with 1 µg/mL of OGNs (red) and IOPs (green) for 1, 2, and 4 h. Yellow color is indicative of the presence of both types of nanoparticles within a particular voxel. Scale bar: 5 µm. (B) Co-localization analysis shows that OGNs were partially co- localized with IOPs initially, but the degree of co-localization decreases with incubation time. On the basis of independent measurements of a set of 10 cells, the threshold Mander’s co-localization coefficients (tM) for the OGNs were computed as 61 ± 10, 46 ± 13, and 45 ± 13% at 1, 2, and 4 h respectively, whereas those for the IOPs were 41 ± 9, 35 ± 12, and 34 ± 10% for 1, 2, and 4 h, respectively. Adapted from [4].

Our research efforts in this direction are motivated by recent work in circulating cell-free cancer biomarker detection using surface enhanced Raman spectroscopy (SERS) assay. We have reported a SERS assay for concomitant detection of three breast tumor antigens (CA 15-3, CA 27-29 and CEA) in ~2 µL of serum with higher sensitivity than existing immunoassays [3]. We developed a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging by functionalizing both the SERS tags and the chip with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enabled the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provided higher sensitivity than conventional immunoassays and demonstrated exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We have further developed a wide-area, compact Raman scanner that can sample the SERS chip in a fraction of the time of standard chemical imaging. Leveraging these developments, we are developing a multiplexed SERS platform for detection of gene-specific hypermethylation through reproducible aggregation of nanoparticles.

Recently, we have also studied the interaction of nanoparticles with cells to understand the fundamental aspects of their cellular uptake, trafficking and gene regulation, particularly in the presence of multiple types of nanoparticles [4]. We employed similarly sized oligonucleotide-modified gold nanoparticles (OGN) and iron oxide nanoparticles (ION) to understand their competitive uptake by HeLa cells upon co-incubation. Our results suggested the minimal influence of each particle on the uptake of the other and hinted at the presence of different uptake pathways for both the nanoparticle types, that can be attributed to factors such as composition and surface properties. Our studies also revealed the fates of both the nanoparticles inside the cells at different time points and their effects on the gene regulation. We will use the insights obtained in these studies to guide precise and site-specific delivery of these cooperative nanoparticles for diverse theranostic applications.

References

  1. "Shedding light on the extinction‐enhancement duality in gold nanostar‐enhanced raman spectroscopy," Angewandte Chemie International Edition, 53(51), 14115-14119 (2014). [ Pubmed ]
  2. "Composite‐scattering plasmonic nanoprobes for label‐free, quantitative biomolecular sensing," Small, 15(38), 1901165 (2019). [ Pubmed ]
  3. "Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay," Chemical Science, 6(7), 3906-3914 (2015). [ Pubmed ]
  4. "Decoding live-cell interactions with multi-nanoparticle systems: differential implications for uptake, trafficking and gene regulation," ACS applied materials & interfaces, 11(37), 33659-33666 (2019). [ Pubmed ]