Multiplexed SERS Detection of Soluble Cancer Protein Biomarkers with Gold–Silver Alloy Nanoboxes and Nanoyeast Single-Chain Variable Fragments

Abstract

Highly sensitive, multiplexed detection of soluble cancer protein biomarkers can facilitate early cancer screening as well as enable real-time monitoring of patients' sensitivity and resistance to therapy. Current technologies for detection of soluble cancer protein biomarkers, e.g., enzyme-linked immunosorbent assay, however, suffer from limited sensitivity, as well as the requirement of expensive monoclonal antibodies, which undergo the quality variability. Herein, we propose a sensitive, cheap, and robust surface-enhanced Raman scattering technology to detect a panel of soluble cancer protein biomarkers, including soluble programmed death 1 (sPD-1), soluble programmed death-ligand 1 (sPD-L1) and soluble epithermal growth factor receptor (sEGFR), which are related to disease progression and treatment efficacy. In this assay, gold-silver alloy nanoboxes that have strong Raman signal enhancement capability were used as plasmonic nanostructures to facilitate highly sensitive detection. In addition, nanoyeast single-chain variable fragments were utilized as mAb alternatives to allow specific and stable protein capture performance. We successfully detected sPD-1, sPD-L1, and sEGFR with a limit of detection of 6.17 pg/mL, 0.68 pg/mL, and 69.86 pg/mL, respectively. We further tested the detection of these three soluble cancer protein biomarkers in human serum and achieved recovery rates between 82.99% and 101.67%. We believe our novel platform that achieves sensitive, multiplexed, and specific detection of soluble cancer protein biomarkers could greatly benefit cancer treatment and improve patient outcome.

Engineering State‐of‐the‐Art Plasmonic Nanomaterials for SERS‐Based Clinical Liquid Biopsy Applications

“…Cyt C (5-10 × 10 −9 m) • SERS substrates for direct SERS detection • SERS nanotags for barcoding Nanodot-decorated gold-silver alloy nanoboxes [38,39] Template-free method EF ≈ 10 6 sPD-1 (6.17 pg mL −1 ), sPDL-1 (0.68 pg mL −1 ), sEGFR (69.86 pg mL −1 )…”

“…Generally, SERS-based protein detection could be performed via i) target-triggered nanoassembly assays; ii) 2D substrate-based immunoassays; [108] iii) pull-down immunoassays; [39,109] and Figure 17. Generally, SERS-based protein detection could be performed via i) target-triggered nanoassembly assays; ii) 2D substrate-based immunoassays; [108] iii) pull-down immunoassays; [39,109] and Figure 17.…”

“…He et al demonstrated an ultrasensitive pull-down immunoassay of detecting N-terminal probrain natriuretic peptide by using metal-organic frameworks embedded with gold tetrapods as SERS substrates. [39] They successfully detected soluble programmed death 1 (sPD-1), programmed death-ligand 1 (sPDL-1), and epithermal growth factor receptor (sEGFR) with LODs of 6.17, 0.68, and 69.86 pg mL −1 , respectively. The proposed SERS immunosensor achieved a large dynamic range of six orders of magnitude from 1 fg mL −1 to 1 ng mL −1 with an LOD of 0.75 fg mL −1 .…”

A SERS microfluidic platform for targeting multiple soluble immune checkpoints

“…PD1, PDL-1 and LAG-3 whole yeast scFv and nanoyeast-scFv were produced as per the method detailed in this work (Li et al 2018). To check the specificity of NY-scFvs, we performed ELISA and found that all the target scFvs have high affinities towards the target antigen (supplementary figure S3).…”

“…These were key two features in achieving detection limits of 100 fg/mL of protein from complex biological sample. SERS PD-1: 6.17 pg/mL PD-L1: 0.68 pg/mL EGFR: 69.86 pg/mL PD-1: 0.01 -1.0 ng/mL PD-L1: 0.001 -10 ng/mL EGFR: 0.1 -1000 ng/mL (Li et al 2018) SERSac-EHD (chip) PD-1: 100 fg/mL PD-L1: 50 fg/mL LAG-3: 100fg/mL PD-1: 100 fg/mL -1ng/mL PD-L1:50 fg/mL -100pg/mL LAG-3: 100 fg/mL -1ng/mL Our work considered as they are less sensitive and require high concentrations of protein. As shown in Table 1, our method has similar sensitivity like ELISA, however, has better dynamic range with low detection limit up to 100fg/mL.…”