Photonic technologies for liquid biopsies: recent advances and open research challenges

Abstract

The recent development of sophisticated techniques capable of detecting extremely low concentrations of circulating tumor biomarkers in accessible body fluids, such as blood or urine, could contribute to a paradigm shift in cancer diagnosis and treatment. By applying such techniques, clinicians can carry out liquid biopsies, providing information on tumor presence, evolution, and response to therapy. The implementation of biosensing platforms for liquid biopsies is particularly complex because this application domain demands high selectivity/specificity and challenging limit-of-detection (LoD) values. The interest in photonics as an enabling technology for liquid biopsies is growing owing to the well-known advantages of photonic biosensors over competing technologies in terms of compactness, immunity to external disturbance, and ultra-high spatial resolution. Some encouraging experimental results in the field of photonic devices and systems for liquid biopsy have already been achieved by using fluorescent labels and label-free techniques and by exploiting super-resolution microscopy, surface plasmon resonance, surface-enhanced Raman scattering, and whispering gallery mode resonators. This paper critically reviews the current state-of-the-art, starting from the requirements imposed by the detection of the most common circulating biomarkers. Open research challenges are considered together with competing technologies, and the most promising paths of improvement are discussed for future applications.

Keywords: liquid biopsies; oncology; optical microscopy; plasmonics; whispering gallery modes.

Figure 1.

Circulating biomarkers as liquid biopsy for precision medicine. In a blood sample, different complementary circulating biomarkers can be detected, isolated and characterized. This figure summarizes the bioanalytical techniques of tumor-educated platelets, circulating tumor cells, circulating cell-free tumor DNA, extracellular vesicles (e.g. exosomes) and cell-free RNA (e.g., (i) noncoding RNA with miRNA, snRNA & snoRNA and lncRNA and (ii) messenger RNA), cell-free proteins as well as their clinical relevance. Abbreviations: TEP, tumor educated platelet; CTC, circulating tumor cell; ctDNA, circulating tumor DNA; cfRNA, cell-free RNA; cfmiRNA, cell-free microRNA; snRNA, small nuclear RNA; lncRNA, long noncoding RNA; mRNA, messenger RNA; RNA-Seq, RNA sequencing; EpCAM, Epithelial Cell Adhesion Molecule; CK, cytokeratin; RT-qPCR, reverse-transcription-quantitative polymerase chain reaction; EGFR, epithelial growth factor receptor; KRAS, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; AR, androgen receptor; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; ESR1, estrogen receptor 1; ALK, anaplastic lymphoma kinase; PD-L1, programmed death-ligand 1; HER2, Human Epidermal Growth Factor Receptor-2; HR, hormone receptor; AR-V7, androgen receptor splice variant 7; PSMA, prostate-specific membrane antigen; Safe-Seqs, Safe-sequencing system; CAPP-Seq, cancer personalized profiling by deep sequencing; TAM-Seq, tagged-amplicon deep sequencing; CNA, copy number aberrations; WGS, whole genome sequencing; WES, whole exome sequencing.

Figure 2.

Schematic descriptions of several advanced forms of high-resolution microscopy. A) PALM/STORM microscopy: fluorophores are excited by a laser beam hitting the cover glass surface near or beyond (shown here) the angle of total internal reflection. The resulting spots in the fluorescence image can be attributed to single fluorescent molecules, which are localized with approx. 10 nm accuracy. B) SOFI: fluctuating signals from molecules are pixel-wise correlated and cross-correlated, resulting in a higher-resolution image. C) STED: a tightly focused excitation beam is superposed with a shaped deexcitation beam, resulting in higher resolution images by raster-scanning of the beams. D) SIM: an interference pattern is rotated and shifted across the sample. Analysis in Fourier space allows for 2x higher image resolution by advanced mathematical reconstruction algorithms. E) Bessel-beam microscopy: a highly focused, elongated excitation beam is swept across the sample, resulting in an extremely thin light sheet. Fluorescence is collected by a second microscopy objective lens held at 90° with respect to the beam. F) Lattice-light sheet microscopy: multiple elongated beams interfere with each other resulting in a lattice-like excitation pattern.

Figure 3.

Localization microscopy in combination with holographic optical tweezers (HOT). A) Schematics of a combined localization microscopy and HOT setup, where a spatial light modulator (SLM) is used to create multiple optical traps with a near-infrared (IR) laser beam to immobilize non-adherent cells, such as T cells. Excitation of fluorescently labeled samples is achieved e.g. by STORM-like exciation with a visible (VIS) laser beam. B) White-light transmitted (upper image) and STORM image (lower image) of single trapped T cells infected with the human immunodeficiency virus (HIV-1), which is labeled by immunofluorescence.

Figure 4.

SPR and SPRi platforms for liquid biopsy. A) Magnetic nanoparticle-enhanced grating-coupled (GC) SPR sensor for EVs quantitative detection with the schematic illustration of EVs preincubation with the biotinylated lipid-binding ligand (b-ligand) and streptavidin (SA) coated magnetic nanoparticles (MNPs). SAM: thiol self-assembled monolayer, L: lens, POL: polarizer, BS: beam splitter. Reproduced with permission.^[112] 2017, Royal Society of Chemistry. B) SPRi platform for capturing and detecting exosomes in cell culture supernatant. Reproduced with permission.^[114] 2014, American Chemical Society. C) Antimonene-based SPR sensor for miRNA detection with details on the operating principle and fabrication process including antimonene naosheet assembly on the Au film (I), Gold nanorod-ssDNAs adsorbtion on antimonene nanosheets (II), sensor interaction with miRNA solution (III), and release of the gold nanorod-ssDNA from the antimonene nanosheets. Reproduced with permission.^[120] Copyright Springer Nature, 2019.

Figure 5.

nPLEX chip for label-free exosomes detection with details on exosomes biogenesis involving multivescicular body, MVB (a), electromagnetic field distribution close to the nanoholes (b), nanoholes array imaged by scanning electron microscopy (c), measurement setup (d), spectral shift due to functionalization and interaction with exosomes, surface-adsorbed exosomes (e). Reproduced with permission.^[123] Copyright 2014, Springer Nature.

Figure 6.

SERS-based method for phenotypic profiling of cancer-derived small extracellular vesicles. A) Nanotags preparation. B) SERS nanotags and CD63 antibody-functionalized magnetic beads for molecular phenotype profiling of CD63-positive EVs. Reproduced with permission.^[151] Copyright 2020, American Chemical Society.

Figure 7.

Overview of FLOWER. (a) Artistic rendering of exosomes binding to a microtoroid optical resonator. For sensing experiments, light is evanescently coupled into the microtoroid using an optical fiber. (b) Block diagram of FLOWER. Light is sent from a tunable wavelength laser to both the microtoroid and an auto-balanced photoreceiver. The output of the receiver is multiplied by a dither signal to generate an error signal to determine whether the laser frequency matches the cavity resonance frequency (c) Schematic of the flow cell which is used. The sample cell is open on three sides and fluid is flowed through it. (d) As exosomes bind to the surface of the toroid, the resonance frequency of the toroid shifts. This is monitored via active tracking of the resonance via FLOWER. Reproduced with permission.^[165] Copyright 2015, American Chemical Society.

Figure 8.

Exosome binding curves. Blood was taken from the tail vein of a mouse with implanted human tumor cells. Serum samples were flowed over the toroid. The resonance frequency of the microtoroid shifts in response to exosome binding. Samples from later weeks generate a larger shift, presumably due to a greater exosome concentration. The red dashed lines represent an exponential fit to the data. Reproduced with permission.^[165] Copyright 2015, American Chemical Society.

Figure 9.

iMEX platform with a schematic illustration of its components (A), a diagram of the readout circuit (B), and a rendering of the packaged device (C). Reproduced with permission.^[214] Copyright 2016, American Chemical Society.