Multiplexed SERS Detection of Soluble Cancer Protein Biomarkers

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Multiplexed SERS Detection of Soluble Cancer Protein Biomarkers with GoldSilver Alloy Nanoboxes and Nanoyeast Single-Chain Variable Fragments Junrong Li, Jing Wang, Yadveer Singh Grewal, Christopher B. Howard, Lyndon J. Raftery, Stephen M. Mahler, Yuling Wang, and Matt Trau Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02216 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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Analytical Chemistry

Multiplexed SERS Detection of Soluble Cancer Protein Biomarkers with Gold-Silver Alloy Nanoboxes and Nanoyeast Single-Chain Variable Fragments Junrong Li,†,ǁ Jing Wang,†,ǁ Yadveer S. Grewal,† Christopher B. Howard,‽ Lyndon J. Raftery,‽ Stephen Mahler,‽ Yuling Wang,*,‡ and Matt Trau*,†,§ †

Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia. Email: [email protected] ‽ Centre for Advanced Imaging, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia ‡ Department of Molecular Sciences, ARC Centre of Excellence for Nanoscale BioPhotonics, Faculty of Science and Engineering, Macquarie University, Sydney, NSW 2109, Australia. Email: [email protected] § School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia 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, however, suffer from limited sensitivity e.g., enzyme-linked immunosorbent assay (ELISA), as well as the requirement of expensive monoclonal antibodies (mAbs), which undergo the quality variability. Herein, we propose a sensitive, cheap and robust surface-enhanced Raman scattering (SERS) 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 (Au)-silver (Ag) alloy nanoboxes (NBs) that have strong Raman signal enhancement capability were used as plasmonic nanostructures to facilitate highly sensitive detection. In addition, nanoyeast single-chain variable fragments (scFvs) 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 (LOD) 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%-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.

Soluble programmed death 1 (sPD-1), soluble programmed death-ligand 1 (sPD-L1), and soluble epithermal growth factor receptor (sEGFR) exist in human blood and are promising biomarkers for the prediction of patient health and monitoring therapy efficiency.1 Typically, increased sPD-1 is associated with prolonged progression-free survival during erlotinib treatment.2 sPD-L1 is a negative therapeutic and prognostic biomarker in renal cell carcinoma.3 Low baseline sEGFR is related to reduced survival in advanced non-small cell lung cancer.4 As such, there is a significant clinical opportunity to utilize sensitive and multiplexed testing to measure these soluble protein biomarkers for cancer therapy prediction, prognosis, and disease monitoring. However, current technologies for soluble protein detection, e.g., enzyme-linked immunosorbent assay (ELISA), suffer from limitations, including: (1) limited sensitivity;5 and (2) reliance on highly specific affinity reagents, typically monoclonal antibodies (mAbs), which are expensive and exhibit variations in quality.6,7

Surface-enhanced Raman scattering (SERS) is an ultrasensitive spectroscopic technique that can reach single molecule detection sensitivity in some conditions (e.g. molecules located in the ‘hot-spots’).8 Furthermore, this technique is intrinsically suitable for multiplexed detection due to the unique and well-separated SERS spectral peaks.9 Currently, SERS technology has been widely applied for sensitive and multiplexed detection of nucleic acids, plant pathogens, and protein biomarkers, typically by utilizing spherical silver (Ag) and gold (Au) nanoparticles (NPs) as SERS substrates.10-12 Spherical NPs, however, suffer from relatively weak Raman signal enhancement,13 which undermines ultrasensitive detection. In contrast, anisotropic plasmonic nanostructures possess stronger signal enhancement capability due to the enhanced electromagnetic fields on the tips or corners.14 Therefore, we developed anisotropic Au-Ag alloy nanoboxes (NBs) as SERS substrates for ultrasensitive measurements of soluble cancer protein biomarkers.

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Single-chain variable fragments (scFvs) have been recognized as mAb alternatives,15 which can be cheaply and rapidly selected from yeast libraries.16 However, scFvs have the potential to lose their activity when the detection environment differs from the selection environment.17,18 To address this limitation, our group has developed nanoyeast-scFvs wherein the scFvs are stably embedded in fragments of yeast cell walls. In this case, the nanoyeast-scFvs reagents retain the same configuration in both the selection and detection environments.16,19 Nanoyeast-scFvs have been successfully applied to detect recombinant Entamoeba histolytica pathogen antigens.15,16,20 In this study, we further extend the utility of nanoyeast-scFvs as mAb alternatives by combining them with novel Au-Ag alloy NBs to sensitively detect clinically-relevant cancer biomarkers. Specifically, we develop a sandwich-type SERS platform for multiplexed detection of soluble cancer protein biomarkers (sPD-1, sPD-L1 and sEGFR) using highly sensitive Au-Ag alloy NBs as SERS substrates and specific nanoyeast-scFv as affinity reagents. Our assay exhibits two distinct advantages over conventional detection methods: 1) anisotropic Au-Ag alloy NBs exhibit highly bright and stable SERS signals, which are fundamental for sensitive, quantitative and reliable detection; 2) nanoyeast-scFvs demonstrate favorable stability profiles whilst simultaneously reduce manufacturing cost and time.

vettes using the Bio-Rad Gene Pulser XCell. 1mL of YPD medium was added to cells and incubated for 30 min at 30 ˚C. Cells were plated out on SDCAA agar to select for EBY100 cells transformed with scFv gene+PCTCON-2. Plates were incubated for 3-4 days at 30 ˚C and single colonies were then picked and inoculated into 5 mL of SDCAA medium for 20-24 hr at 30 ˚C. The culture was diluted in SGCAA induction medium to optical density of 1.0 at 600 nm in a final volume of 10 mL and induced at 20 ˚C for 48 hr. Expression of scFvs in the yeast was determined by immunoblot via detection of the c-myc epitope tag at the C-terminal of the scFvs. For immunoblot, 10 µL of non-induced (SDCAA) and induced (SGCAA) samples were run on polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes, and the presence of expressed scFvs was probed with HRP labelled anti-c-myc antibody. Prior to use in an assay, whole yeast-scFvs in growth media were centrifuged at 13000 g for 2 min at room temperature to pellet whole yeast-sFvs. Supernatant was removed and the pellet was resuspended in 10 mL of PBS, 5% glycerol, and protease inhibitor EDTA-free cocktail. To this solution of whole yeast-scFvs in PBS/glycerol, several 3 mm ball bearings were added into the sample and vortexed for 15 s to fragment the whole yeast-scFvs into nanoyeast fragments in solution. The solution was then filtered using a 0.1 µm filter to obtain only sub-100 nm sized fragments. For storage, 0.05% sodium azide was added to prevent microbial growth. Synthesize of Au-Ag alloy NBs. In a typical synthesis of 80 nm NBs, 45 µL of 25.4 mM HAuCl4 was added into 10 mL of H2O under magnetic stirring. After that, 170 µL of 6 mM AgNO3 and 30 µL of 0.1 M AA were introduced into the solution simultaneously. Obvious blue color can be observed immediately, indicating the formation of the NBs. The mixture was kept stirring for 1 min. Then, the final product was collected by centrifugation at 600 g for 15 min and resuspended into H2O for characterization. Preparation of SERS nanotags. SERS nanotags were prepared by forming a mixed thiol monolayer composed of Raman reporters and DSP, followed by conjugation of antibodies via DSP molecules. Briefly, 8 µL of 1 mM MBA, 10 µL of 1 mM DTNB and 10 µL of 1 mM TFMBA were added with 2 µL of 1 mM DSP into 300 µL of Au-Ag alloy NBs concentrated from 1 mL of original solution, respectively. The mixtures were incubated at 25 °C, 350 rpm for 6 h to form the intact monolayers. After that, the solutions were centrifuged at 600 g for 15 min to remove the residual reactants and resuspended into 300 µL of 0.1 mM PBS. Next, 2 µL of 1 mg/mL anti-PD-1, anti-PD-L1 and anti-EGFR antibodies were added into the solutions and incubated at 4 °C (refrigerator) overnight for the immobilization of antibodies onto the surfaces of the Au-Ag alloy NBs. Finally, the products were centrifuged at 600 g for 15 min to get rid of free antibodies, and resuspended into 300 µL of 0.1% BSA to prevent nonspecific adsorption and the detaching of Raman reporters. The asprepared SERS nanotags were stored in refrigerator for future use.

EXPERIMENTAL SECTION Chemicals and materials. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), silver nitrate (AgNO3), 4mercaptobenzoic acid (MBA), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), and dithiobis(succinimidyl propionate) (DSP) were purchased from Sigma Aldrich. Ascorbic acid (AA) was obtained from MP Biomedicals, Inc.. Streptavidin coated magnetic beads (MBs) were bought from Thermo Fisher Scientific. Biotin HA antibody was obtained from Sapphire Bioscience. Recombinant PD-1 (Cat# 1086-PD-050), PD-L1 (Cat# 156-B7-100) and EGFR (Cat# 1095-ER) proteins were purchased from R&D Systems. PD-1 (Cat# 329902), PD-L1 (Cat# 329702) antibodies were bought from BioLegend. EGFR antibodies (Cat# mAb1095) were obtained from R&D Systems. All of the above chemicals were analytical reagents and used without further purification. Ultrapure water with an electrical resistance of 18.2 MΩ·cm was used throughout the experiments. Human blood was obtained from the Australian Red Cross Blood Service. Generation of nanoyeast-scFv. Whole yeast scFvs were expressed on the surfaces of the Saccharomyces ceriviseae EBY100 strain using a modified version of Van Deventer and Wittrup.21 To do this, genes encoding scFvs that target PD-1, PD-L1 and EGFR protein biomarkers were synthesized by Geneart (Invitrogen) and cloned into the pCTCON-2 yeast display gene vector using restriction enzymes Nhe1 and Sal1, and T4 ligase (NE Biolabs). 700-1000 ng of DNA (10 µL of scFv genes+pCTCON-2) was transformed into 250 µL of electrocompetent EBY100 (treated with 100 mM lithium acetate and 10 mM 1,4-dithiothreitol), using electroporation with a square wave protocol of 1 pulse at 500V/15ms in 2 mm cu-

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Analytical Chemistry

Figure 1. Au-Ag alloy NB characterizations for the size, structure, elemental distribution and optical property. (A) TEM image; (B) TEM image for a single NB; (C) HR-TEM image taken from the red circle in B; (D) EDS mapping for Au; (E) EDS mapping for Ag; (F) Mixed EDS mapping for Au and Ag; (G) Cross-section EDS line profile of the NB in figure B, the inset shows the signal collecting direction; (H) UV-vis extinction spectrum of the NBs.

into the mixture and incubated at 37 °C, 850 rpm for 30 min. After removing the free soluble cancer protein biomarkers, PD-1, PD-L1 and EGFR SERS nanotags were added and incubated at 37 °C, 850 rpm for 30 min. Finally, the free SERS nanotags were washed away with 0.1% BSA-0.01% Tween 20 three times and resuspended into 60 µL of H2O for SERS measurements. Recovery test and multiplexed detection of sPD-1, sPDL1 and sEGFR in human serum. For the recovery test, the human serum was diluted 100-fold using PBS. Then, different amounts of sPD-1, sPD-L1 and sEGFR were added into the serum to the final concentration of 1 ng/mL, 0.1 ng/ mL and 10 ng/mL, respectively. Next, 100 µL of serum was detected in a similar way in PBS. For the detection of multiple soluble cancer protein biomarkers in serum, the different combinations of soluble cancer protein biomarkers were added into the 100 µL of 100-fold diluted serum, followed by incubating with the mixture of PD1, PD-L1, and EGFR nanoyeast-scFvs functionalized MBs at 37 °C, 850 rpm for 30 min. The free soluble cancer protein biomarkers were removed using magnetic separation. Then, PD-1, PD-L1, and EGFR SERS nanotags were incubated with the solutions at 37 °C, 850 rpm for 30 min, and washed with 0.1% BSA-0.01% Tween 20 three times to get rid of the free SERS nanotags. Finally, the products were resuspended into 60 µL of H2O to conduct SERS measurements. Instrumentation. The transmission electron microscope (TEM) image was taken on a Hitachi HT7700 microscope (Hitachi, Tokyo, Japan) operated at 120 kV. High resolution (HR)-TEM and energy dispersive X-ray spectroscopy (EDS) images were performed on a JEOL-2100 microscope with an

Sensitive detection of sPD-1, sPD-Ll and sEGFR in PBS. The SERS detection of these soluble cancer protein biomarkers was based on the magnetically assisted sandwichtype structure. For sPD-1 detection, 5 µL of streptavidin coated MBs were transferred into 2.0 mL of Eppendorf tubes and washed with PBS. Then, 0.5 µL of 1 mg/mL biotin-HA antibodies were added into the washed MBs and incubated at 25 °C, 850 rpm for 30 min. The free biotin-HA antibodies were removed by magnetic separation. Next, 10 µL of PD-1 nanoyeast-scFvs were added into the solution and incubated at 25 °C, 850 rpm for 30 min, followed by discarding the unbounded nanoyeast-scFvs with magnetic separation. After that, the MBs were blocked with 100 µL of 1% BSA to prevent nonspecific binding. Then, different amounts of sPD-1 proteins were incubated with the PD-1 nanoyeast-scFvs functionalized MBs at 37 °C, 850 rpm for 30 min and the free sPD-l proteins were removed after that. 20 µL of PD-1 SERS nanotags were added into the system to bind with the sPD-1 proteins by incubating at 37 °C, 850 rpm for 30 min. The free and nonspecific adsorbed PD-1 SERS nanotags were washed away with 0.1% BSA-0.01% Tween 20 three times and concentrated into 60 µL of H2O for Raman interrogation. The detection of sPD-L1 and sEGFR can be realized by replacing the nanoyeast-scFvs, target protein and corresponding SERS nanotags into those that can match sPD-L1 and sEGFR, respectively. It is worth noting that each sample was prepared in triplicates. For the detection of multiple soluble cancer protein biomarkers in PBS, PD-1, PD-L1, and EGFR nanoyeast-scFvs functionalized MBs were mixed firstly. Then, the combination of all possible soluble cancer protein biomarkers were added

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Scheme 1. Schematic illustration for the detection of sPD-1, sPD-L1 and sEGFR. accelerating voltage at 200 kV. UV-vis extinction spectrum was recorded on a UV-2450 UV-vis spectrophotometer (Shimadzu). Particle size distribution (PSD) profile was measured by DCS disc centrifuge (model 24000UHR) CPS Instruments Inc.. The disc was loaded with 14.4 mL of sucrose gradient fluid containing 8-24% sucrose in H2O. Scanning electron microscope (SEM) images were obtained with a JEOL-7100 FE-SEM microscope with 20 kV voltage. Raman measurements were collected from a portable IM-52 Raman microscope that provided a 70 mW laser excitation at 785 nm.

enriched NBs exhibits a distinct advantage over the traditional NBs that have a significant loss of Ag.26 UV-vis extinction spectroscopy was utilized to explore the optical property of the NBs, where an obvious surface plasmon resonance (SPR) peak was observed at 610 nm (Figure 1H). This red shifted SPR peak, relative to that of solid spherical AuNPs and AgNPs, enables the NBs to act as superior SERS substrates when using long wavelength laser excitation (e.g. 632.8 nm and 785 nm), because the resonance frequency of the NBs is in larger resonance with the laser radiation.8 We then compared the SERS enhancement capability between Au-Ag alloy NBs and 60 nm AuNPs, considering that 60-80 nm AuNPs possess the strongest SERS enhancement capability in spherical AuNPs.8 Au-Ag alloy NBs and 60 nm AuNPs under the same concentration were functionalized with MBA as Raman reporters to realize SERS enhancement comparison. As shown in Figure S1, Au-Ag alloy NBs generate almost 6 times stronger signals than AuNPs, mainly due to the “hot spots” on the sharp corners and the retainment of Ag. Given that Au-Ag alloy NBs exhibit higher SERS activity, we thus applied Au-Ag alloy NBs for sensitive detection of soluble cancer protein biomarkers. Detection principle of multiple soluble protein biomarkers. Scheme 1 shows the principle of the SERS platform for sPD-1, sPD-L1 and sEGFR detection utilizing Au-Ag alloy NBs as plasmonic nanostructures and nanoyeast-scFvs as affinity reagents. Briefly, biotin-HA antibodies are first attached to streptavidin-coated magnetic beads (MBs), followed by conjugating nanoyeast-scFvs to MBs through the specific interactions between HA antibodies and HA antigen tags cloned into the recombinant scFv construct. Nanoyeast-scFvs are defined based on their sizes in the nanometer range, which have been comprehensively characterized in our previous work.27 The nanoyeast-scFvs functionalized MBs are subsequently applied to capture these soluble cancer protein biomarkers. SERS nanotags, composed of polyclonal antibodyconjugated and Raman reporter-coated Au-Ag alloy NBs, are then used for identification and quantification of the captured soluble cancer protein biomarkers. The formation of nanoassemblies (NBs on the surfaces of magnetic beads) (Figure S2)

RESULTS AND DISCUSSION Highly sensitive Au-Ag alloy NBs as SERS plasmonic nanostructures. We synthesized anisotropic Au-Ag alloy NBs by reducing HAuCl4 and AgNO3 with AA. This protocol follows a straightforward and environmentally friendly synthesis workflow and does not require surfactants, high temperature or an organic phase. The prepared NBs have an average edge length of 80 nm as shown in TEM images (Figure 1A and B), which is within the ideal SERS plasmonic nanostructure size (