Recent Progress on Liquid Biopsy Analysis using Surface-Enhanced Raman Spectroscopy

Abstract

Traditional tissue biopsy is limited in understanding heterogeneity and dynamic evolution of tumors. Instead, analyzing circulating cancer markers in various body fluids, commonly referred to as "liquid biopsy", has recently attracted remarkable interest for their great potential to be applied in non-invasive early cancer screening, tumor progression monitoring and therapy response assessment. Among the various approaches developed for liquid biopsy analysis, surface-enhanced Raman spectroscopy (SERS) has emerged as one of the most powerful techniques based on its high sensitivity, specificity, tremendous spectral multiplexing capacity for simultaneous target detection, as well as its unique capability for obtaining intrinsic fingerprint spectra of biomolecules. In this review, we will first briefly explain the mechanism of SERS, and then introduce recently reported SERS-based techniques for detection of circulating cancer markers including circulating tumor cells, exosomes, circulating tumor DNAs, microRNAs and cancer-related proteins. Cancer diagnosis based on SERS analysis of bulk body fluids will also be included. In the end, we will summarize the "state of the art" technologies of SERS-based platforms and discuss the challenges of translating them into clinical settings.

Keywords: circulating tumor DNA; circulating tumor cell; exosome; liquid biopsy; surface-enhanced Raman spectroscopy.

Figure 1

Circulating biomarkers in different body fluids, including blood, urine, saliva, ascites, cerebrospinal fluid, etc. Adapted with permission from , copyright 2017 BMC.

Figure 2

(A) Illustration of the collective oscillation of free electrons in metal nanoparticles upon excitation by an electromagnetic wave. (B) Illustration of electromagnetic enhancement and chemical enhancement in SERS.

Figure 3

Representative SERS substrates. (A) Nanostructured surfaces prepared by controlled deposition of NPs or lithographic/template synthesis methods. (B) Colloidal NPs-based SERS substrates, including quasi-spherical NPs, anisotropic NPs, aggregates of NPs and NP assemblies.

Figure 4

SERS-based liquid biopsy analysis using a label-free SERS approach (left) or SERS tags (right). In label-free SERS, the spectroscopic signal results from analyte adsorption onto the SERS substrate, whereas in SERS tags-based specific recognition assays, the spectroscopic signal results from the reporter molecules on the SERS tags.

Figure 5

SERS detection of CTCs after magnetic beads enrichment. (A) a, Schematic of the ternary immuno-complex formed by SERS tags and magnetic bead conjugates binding to the model CTC. b, TEM image of the SERS tags. c, Magnetic beads binding to SKBR3 cells. d, HER2 antibody-conjugated SERS tags (green) labeling of the SKBR3 membrane (Hoechst dye-labeled nuclei are in blue). Adapted with permission from , copyright 2008 American Chemical Society. (B) Schematic representation of CTCs capture and identification using aptamer-modified magnetic beads and SERS tags. Adapted with permission from , copyright 2015 Springer Nature.

Figure 6

Schematic of the ASGPR antibody-conjugated silver‐coated magnetic NPs, GPC3 antibody-conjugated SERS tags, and the operating principle for CTCs detection in human peripheral blood using dual-enhanced SERS.

Figure 7

Schematic of the Raman-encoded, PEG-stabilized, and EGF peptide-functionalized SERS tags and the assay principle for their use in CTCs detection in human peripheral blood without enrichment.

Figure 8

(A) Schematic of SERS-active NPs with various shapes for CTCs detection. (B) TEM images of Au nanospheres-based SERS tags, Au nanorods-based SERS tags, Au nanostars-based SERS tags, and their sensitivity for CTCs detection. Adapted with permission from , copyright 2016 American Chemical Society.

Figure 9

Detection of multiple surface markers on CTCs using SERS. (A) Schematic and Raman spectra of four antibody-modified SERS nanotags. (B) Schematic of breast cancer cell surface targeting by four SERS tags and a SERS/photothermal detection technique. (C) Schematic of 2D multi-color SERS data correlation with SERS tag' distribution on the cell surface. (D) Multicolor SERS analysis of a single MCF-7 cell among WBCs (d1), a single MCF-7 cell in whole blood (d2), and WBCs only (d3). Adapted with permission from , copyright 2014 Springer Nature.

Figure 10

(A) Experimental workflow for the use of four melanoma surface marker antibodies (MCSP, MCAM, ErbB3, and LNGFR)-modified SERS tags to monitor CTC surface marker expression. (B) CTC populations in response to treatment: the frequency distribution of each marker can signal how diverse the cell populations are in terms of surface marker expression levels. (C) CTC signature in response to treatment: the relationship between the average Raman intensities of each surface marker represents the CTC signature. Adapted with permission from , copyright 2018 Springer Nature.

Figure 11

Capture, detection and release of CTCs. (A) Schematic of the preparation of a supersensitive CTC analysis system based on Ag nanoprisms and SPION (a), and its application to the capture, enrichment, detection, and release of CTCs (b). Adapted with permission from , copyright 2018 American Chemical Society. (B) Schematic of the selective detection and analysis of CTCs and circulating cancer stem cells for monitoring tumorigenesis and metastasis.

Figure 12

Schematic of the preparation of aptamer-modified magnetic beads, SERS tags and the work flow of SERS-based detection of exosomes. Adapted with permission from , copyright 2018 The Royal Society of Chemistry.

Figure 13

(A) Experimental workflow of the fabrication of an antibody array and the detection of exosomes by the antibody array and SERS tags. (B) Average SERS spectra of exosomes at different concentrations captured with CD63 antibodies, average SERS spectra from exosomes using different capture antibodies, and a colorimetric comparison of protein expressions on cancer and normal cells based on SERS detection results. Adapted with permission from , copyright 2018 Ivyspring.

Figure 14

Label-free SERS detection of exosomes. (A) Schematic of a silver film-coated nanobowl substrate preparation and its use in SERS analysis of intact and ruptured exosomes (a). Time-dependent SERS spectra of exosomes derived from the SKOV3 cell line (b) and principal component analysis of the SERS spectra (c). Adapted with permission from , copyright 2015 The Royal Society of Chemistry. (B) Schematic of exosomes released from lung cancer cells (a) and normal cells (b), and SERS detection of the two types of exosomes (c, d). e, SERS spectra of exosomes released from lung cancer cells (blue) and normal cells (black). f, Exosome classification by PCA of SERS spectra. Adapted with permission from , copyright 2017 American Chemical Society.

Figure 15

SERS-based detection of circulating tumor DNA. (A) Schematic of SWNT-based SERS assay coupling with RNase HII-assisted amplification for highly sensitive detection of ctDNA in human blood. Adapted with permission from , copyright 2016 American Chemical Society. (B) Schematic of a multiplexed PCR/SERS assay: multiplex mutation-specific primers were used to amplify tumor DNA, the amplicons were then tagged with mutation-specific SERS nanotags and enriched using magnetic beads. After that, Raman detection was performed for evaluation of the mutation based on the corresponding unique spectral peaks. Adapted with permission from , copyright 2016 Ivyspring.

Figure 16

(A) Schematic of the concept of “Click SERS” analogous to click chemistry. (B) a, Schematic of a 15-well plate for 10-plex DNA detection and top view of the detection wells with full spectra when 10 targets are added. b, TEM images of dimer formation. c, Acquired “Click” spectra presented in the wells during sample detection. Adapted with permission from , copyright 2018 American Chemical Society.

Figure 17

SERS-based detection of circulating microRNA. (A) Schematic of the synthesis of mushroom-like Au-Ag SERS probes by either using a Raman reporter-labeled alkanethiol probe DNA (1), or co-assembling thiol-containing Raman reporter molecules with the probe DNA (2), and formation of the sandwich complexes by hybridization of target DNA/RNA with capture beads and SERS probes. Adapted with permission from , copyright 2017 American Chemical Society. (B) Schematic of the preparation and application of the molecular beacon-functionalized SERS sensor (signal turn “on/off”) for simultaneously measuring multiple miRNAs. Adapted with permission from , copyright 2016 The Royal Society of Chemistry. (C) Design scheme of enzyme-free quadratic SERS signal amplification for circulating microRNA detection in human serum via miRNA-triggered hybridization chain reaction and Ag⁺-mediated cascade amplification. Adapted with permission from , copyright 2015 The Royal Society of Chemistry.

Figure 18

SERS-based detection of PSA. (A) Schematic of a SERS-based assay for the simultaneous detection of f-PSA and c-PSA: (i) mixing of f-PSA, c-PSA, and t-PSA antibody-conjugated magnetic beads; (ii) addition of SERS nanotags to form sandwich immunocomplexes; (iii) separation of magnetic immunocomplexes using a magnetic bar; simultaneous detection of (iv) f-PSA and (v) c-PSA. (B) TEM images of magnetic beads before and after the formation of magnetic immunocomplexes at a 5:5 molar ratio of f-PSA and c-PSA. (C) Raman spectra of (i) f-PSA antibody/MGITC-labeled AuNPs, (ii) c-PSA antibody/XRITC-labeled AuNPs and (iii) their 1:1 (V/V) mixture. (D) Raman intensity variations for different molar ratios of f-PSA and c-PSA (9:1, 8:2, 7:3, 6:4, and 5:5). Adapted with permission from , copyright 2017 American Chemical Society.

Figure 19

(A) Schematic of HCPCF as a SERS platform. (B) Schematic of the binding of anti-EGFR antibody-conjugated SERS tags to the target proteins immobilized on the inner wall of the core of HCPCF.

Figure 20

(A) Scheme of a SERS sensor for the detection of mucin-1 based on Au NRs-Ag NPs core-satellite assemblies. (B) TEM images of Au NRs-Ag NPs core-satellite assemblies with different concentrations of mucin-1. (C) SERS spectra and standard curve of mucin-1 detection. Adapted with permission from , copyright 2015 The Royal Society of Chemistry.

Figure 21

(A) Schematic of a Ag NPs aggregation-based SERS signal generation system. Adapted with permission from , copyright 2015 American Chemical Society. (B) a, Schematic of SERS tags binding on BMFON after MMP-2/MMP-7 enzyme cleavage of peptides on BMFON and AuNPs. b, SEM images of (i) clean BMFON substrate (ii) peptide-shielded avidin-conjugated BMFON and (iii) SERS tags bound to BMFON after enzyme cleavage. Adapted with permission from , copyright 2015 Optical Society of America.

Figure 22

(A) Schematic of mechanical deformation in an antibody-conjugated 4ATP sensor. Adapted with permission from , copyright 2012 American Chemical Society. (B) Schematic of microcontact printing to define ordered domains of chemisorbed Raman reporters on the substrate (left) and target molecules-induced Raman frequency shift (right). Adapted with permission from , copyright 2016 American Chemical Society. (C) Scheme showing label-free determination of human IgG in blood samples obtained by finger prick. Adapted with permission from , copyright 2014 American Chemical Society.