Urine biopsy technologies: Cancer and beyond

Abstract

Since the discovery of circulating tumor cells in 1869, technological advances in the study of biomarkers from liquid biopsy have made it possible to diagnose disease in a less invasive way. Although blood-based liquid biopsy has been used extensively for the detection of solid tumors and immune diseases, the potential of urine-based liquid biopsy has not been fully explored. Advancements in technologies for the harvesting and analysis of biomarkers are providing new opportunities for the characterization of other disease types. Liquid biopsy markers such as exfoliated bladder cancer cells, cell-free DNA (cfDNA), and exosomes have the potential to change the nature of disease management and care, as they allow a cost-effective and convenient mode of patient monitoring throughout treatment. In this review, we addressed the advancement of research in the field of disease detection for the key liquid biopsy markers such as cancer cells, cfDNA, and exosomes, with an emphasis on urine-based liquid biopsy. First, we highlighted key technologies that were widely available and used extensively for clinical urine sample analysis. Next, we presented recent technological developments in cell and genetic research, with implications for the detection of other types of diseases, besides cancer. We then concluded with some discussions on these areas, emphasizing the role of microfluidics and artificial intelligence in advancing point-of-care applications. We believe that the benefits of urine biopsy provide diagnostic development potential, which will pave opportunities for new ways to guide treatment selections and facilitate precision disease therapies.

Keywords: Liquid biopsy; cell-free DNA; disease monitoring; exfoliated bladder cancer cells; extracellular vesicles.

Figure 1

Lateral flow assays for the detection of biomarkers associated with multiple disease types. (A) The rabbit anti-SP antibodies (primary antibodies) of a lateral flow assay were immobilized on the sample line (orange), and the goat anti-rabbit antibodies (secondary antibodies) were immobilized on the control line (green). Rabbit anti-SP antibodies conjugated with colloidal gold particles were fixed by fibrous support (yellow), but they were mobilized when fluid was introduced from the sample pad . (B) An ELISA assay. (1) The urinary sample was added to the substrate coated with LAM-specific antibodies. (2) Mobilized particles were removed by washing with PBS (3) Horseradish peroxidase (HRP) -conjugated LAM-specific rabbit polyclonal antibodies were applied to the substrate, followed by the removal of all mobilized particles with PBS. (4) Tetramethylbenzidine (TMB) was added to the substrate. (5) The enzymatic reaction between TMB and HPR produced a colorimetric signal.

Figure 2

Microfluidics to facilitate the detection of biomarkers from a urine-based liquid biopsy. (A) In a DLD device, particles move either in a transverse mode (orange) or bumping mode (green), as shown. The diameter of particles and nominal critical diameter is characterized as D_P and D_C, respectively. When D_P < D_C, particles follow the laminar flow in a zigzag mode (θ = 0, orange), while for D_P ≥ D_C, particles follow a bumping mode (θ = θ_max, green). (B) In the nanowire-anchored microfluidic device, untreated urine samples are injected into the inlet of the device, the EVs in urine samples were captured by anchored ZnO nanowires based on electrostatic interactions. At the same time, uncollected urinary free-floating objects are collected in the outlet .

Figure 3

Assays based on the fluorescence-based analysis. (A) In an optical sensor using long persistent phosphors (LPPs) and photonic crystals as a substrate, signal enhancement via photonic crystals substrate minimizes transmitted light, resulting in the enhancement of reflected luminescent signal. The light emitted by the LPPN penetrates the substrate (blue) without photonic crystals but cannot penetrate the substrate in the presence of photonic crystals (grey). (B) In a procedure based on the use of renal-clearable catalytic gold nanoclusters, tumor-associated proteases cleave the peptide linker between the gold nanoparticle complex and neutral avidin. The presence of AuNCs is tested by adding hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) to the urine as AuNCs catalyse the reaction between hydrogen peroxide and TMB. The blue colour produced indicates the presence of AuNCs found in urine (cancer presence).