Monosaccharide-mediated rational synthesis of a universal plasmonic platform with broad spectral fluorescence enhancement for high-sensitivity cancer biomarker analysis | Journal of Nanobiotechnology

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Materials

Gold chloride trihydrate (HAuCl4·3H2O), silver nitrate (AgNO3), cell culture-grade dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. All saccharides including d-glucose, d-mannose, d-xylose, d-galactose, d-maltose, sucrose, and l-arabinose were acquired from Shanghai Macklin Biochemical. Poly-l-lysine (PLL) and horseradish peroxidase-conjugated BSA (HRP-BSA) were obtained from Solarbio. Water-soluble amine-reactive N-hydroxysulfosuccinimidobiotin (sulfo-NHS-biotin) and streptavidin (SA) were bought from APExBIO. CF488A, CF555, and CF647 fluorescent dyes in NHS esters were attained from Biotium. Mouse anti-human PSA monoclonal antibody pairs (clones: 7H2 and 8A12 as capture and detection antibodies, respectively) and recombinant human PSA expressed in an E. coli system were purchased from GenScript. Phosphate-buffered saline (PBS) used in the study was 1 × dilution (10 mM Na2HPO4, 1.75 mM KH2PO4, 137 mM NaCl, and 2.65 mM KCl at pH 7.2–7.6) from a 20 × stock (Sangon Biotech) and autoclaved for sterilization before use unless otherwise noted. 18.2 MΩ-cm ultrapure Milli-Q® water (MilliporeSigma) was used in the entire study.

Characterization

Ultraviolet–visible (UV–vis) absorption spectra were recorded using a UV-2600 spectrophotometer (Shimadzu) in a quartz cuvette or an Infinite M200 Pro multimode microplate reader (Tecan) in a microplate. All fluorescence measurements were conducted using the Tecan plate reader in ultrathin special optics PS microplates with minimal background fluorescence. Morphologies of nanostructures were examined by a JEOL JSM-6700F field emission-scanning electron microscope (FE-SEM) operating at 10 kV. Energy-dispersive X-ray spectroscopy (EDS) and elemental mapping were acquired with a high-resolution Zeiss Gemini500 thermal FE-SEM at 6 kV accelerating voltage. Surface hydrophobicity was indicated by water contact angles measured with a DSA100 Drop Shape Analysis System (Kruss) in ambient conditions. Protein secondary structures were assessed by a Nicolet 6700 Fourier transform infrared (FTIR) spectrometer (Thermo Fisher Scientific) in the attenuated total reflection (ATR) mode. All electrochemical measurements were performed on a CHI 630E Electrochemical Analyzer (CH Instruments) in a standard three-electrode configuration comprised of a glassy carbon working electrode, a Pt plate counter electrode, and a Ag/AgCl reference electrode.

Synthesis of Ag nanoisland substrate

AgNIS was synthesized through a two-step surface-based seed-mediated growth, using a modified silver redox reaction with AuNPs as catalysts and nucleation centers [31]. First, AuNPs were synthesized using a modified Turkevich method [32, 33]. Succinctly, 15 mL 1% w/w sodium citrate was added to 100 mL 1 mM HAuCl4 for 15 min of boiling. After being cooled to room temperature (RT), the reaction mixture was passed through a 0.22 μm membrane filter. Subsequent synthesis of AgNIS was initiated by adding 50 μL preceding citrate-capped AuNPs solution as seeds to PS surface that was pretreated overnight at RT with 50 μL PLL in concentrations ranging from 0.001 to 1 mg mL−1. After 2 h of incubation at RT followed by washing, a freshly prepared Tollens’ reagent containing 24 mM NaOH, 13 mM NH4OH, and 20 mM AgNO3 to form the colorless Ag(NH3)2+ complex. 100 μL Tollens’ reagent at various concentrations was added to the AuNP-seeded surface, followed by 100 μL 10 mM reducing sugars of different kinds. The incubation was kept on a microplate shaker for 15 min for the growth of Ag nanostructures.

Benedict’s assay

To correlate the reducing power of a variety of saccharides with Ag nanostructures and corresponding fluorescence enhancement, we conducted a semi-quantitative assay by preparing Benedict’s reagent constituted of 69 mM CuSO4, 0.95 M Na2CO3, and 0.67 M sodium citrate. 50 mM saccharide was mixed with the above Benedict’s reagent at a v/v ratio of 1:9 and incubated on a thermostatic water bath at 100 °C for 8 min. Absorption was measured at 260 nm, and the non-reducing disaccharide sucrose was used as a negative control.

Determination of fluorescence enhancement

Native BSA was selected as the model molecular probe because its hydrodynamic radius of approximately 7 nm in ambient conditions provides a viable distance between fluorophores and plasmonic surface [34]. BSA was covalently modified with CF488A, CF555, and CF647 by amine-NHS crosslinking. Each fluorophore was dissolved in anhydrous DMSO and added to a pH 8 PBS solution containing 0.1 molar equivalence of BSA, with the final DMSO volume restricted below 0.5% v/v to advantageously reserve native protein structure. The reaction mixture was incubated on a shaker at RT for 2 h. Purification was performed with NAP size-exclusion chromatography columns (Cytiva). The resulting complexes were added to as-synthesized AgNIS for 2 h of incubation at RT and sealed from light until measurement of MEF at different wavelengths. Fluorescence intensities (FIs) were quantified in Quantity One software v4.6.6 (Bio-Rad) by measuring regions of interest (ROIs) of fluorescence images scanned with a Molecular Imager PharosFX Plus System (Bio-Rad) and a home-built microplate adaptor. Background (BKGD) was subtracted, and the enhancement factor was calculated according to the following equation:

$$EF \left( {Enhancement factor} \right) = \frac{{FI\left( {AgNIS} \right) – BKGD\left( {AgNIS} \right)}}{{FI\left( {PS} \right) – BKGD\left( {PS} \right)}}$$

Finite-difference time-domain simulation

Optical simulations were performed with the commercial software FDTD Solutions (Lumerical). The structure of irregularly-shaped semi-continuous AgNIS was established by importing the corresponding SEM image with a selected region of size 2000 nm × 1500 nm on the PS substrate. The refined mesh area with a mesh size of 1 nm was utilized to cover the entire nanostructure to assure simulation accuracy. The dielectric function of Ag is based on the optical constants given by the CRC handbook [35].

Determination of gap distance distribution

The distance distribution of inhomogeneous gaps was quantified by gap skeletonization and computation of gap distances. Firstly, we categorized all image pixels into binary gaps (0) or silver structures (1) through the imbinarize function in MATLAB software R2020b with global thresholding computed using Otsu’s method [20], followed by skeletonization and distance computation. Next, the ‘clean’ operation of the bwmorph function was performed, followed by the ‘majority’ operation to optimize final binary images. The gap skeleton was acquired by inverting binary images with the imcomplement function and applying the 4-connectivity bwskel function to the inverted images to preserve the topology and Euler number of gaps. The gap distance is defined as twice the shortest distance from the medial skeleton line of gaps to the sliver structural boundary. Then, the Euclidean distance transformed by the bwdist function was applied to collect gap distances from each pixel to the nearest sliver boundary. Statistical analyses included only the Euclidean distance of the gap skeleton. Finally, the Distribution Fitter app in MATLAB was used for gamma distribution fitting.

Synthesis of Ag nanoparticles

Ag nanoparticles (AgNPs) were synthesized using a solution-phase approach analogous to AgNIS. Briefly, 1 mL 100 mM mannose aqueous solution was added to 9 mL premixed AgNO3 and NH4OH at final concentrations of 1 mM and 5.9 mM, respectively. 10 mM NaOH was then introduced into the reaction to accelerate the reduction rate. The reaction mixture was maintained at RT for 10 min under stirring at 500 rpm. The morphology was visualized by a JEOL JEM-2010HR transmission emission microscope (TEM) operating at 200 kV, and the hydrodynamic size was measured by Zetasizer Nano ZSE (Malvern Panalytical).

Preparation of continuous Ag thin films by magnetron sputtering

To investigate the influence of structural continuity on fluorescence enhancement, we synthesized a continuous Ag thin film on PS that was pretreated with 0.001 mg mL−1 PLL overnight at RT to increase substrate adhesion. The substrate was placed on the holder at a 20° angle of inclination to ensure successful deposition. Radiofrequency magnetron sputtering deposition was conducted with a silver target in magnetron discharge plasma at the sputtering power of 60 W for 5 min using a VTC-300 system (ZKDS Technology).

Solution-phase synthesis of Au thin films

The PS surface pretreated with 0.001 mg mL-1 PLL was modified by AuNPs seeds identically as AgNIS. After washing three times with water to remove unbound AuNPs, it was immersed in a 10 mM equimolar solution of HAuCl4 precursor and hydroxylamine and placed on a shaker for 15 min at RT to complete the growth process.

Cell lines and culture

Cell lines were all acquired from American Type Culture Collection (ATCC). PC-3 (derived from human prostate adenocarcinoma) and LNCaP (derived from human prostate carcinoma) were cultured in complete Gibco Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Cells were grown in a Heracell VIOS 160i incubator (Thermo Fisher Scientific) with a humidified atmosphere and 5% CO2 at a constant 37 °C. Cell counting was accomplished in PD100 counting chambers using a Cellometer Auto 1000 automated cell counter (Nexcelom Bioscience), following trypan blue staining.

Western blotting

The 1 × cell lysis buffer was prepared by diluting a 10 × radioimmunoprecipitation assay (RIPA; 0.5 M Tris–HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) buffer (MilliporeSigma) supplemented with phosphatase and protease inhibitor cocktails (Bimake). Cells were harvested with cell scrapers and lysed in cell lysis buffer on ice for 30 min. Lysates were cleared by centrifugation at 12,000 g for 20 min, with the supernatant transferred for normalization of concentrations using a BCA protein quantification kit (Pierce). Loading lysates were prepared by adding NuPAGE LDS sample buffer (Invitrogen) and reduced for 10 min at 100 °C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto methanol-activated Immobilon-P PVDF membranes (MilliporeSigma) at 210 mA constant current for 1 h. The blotted PVDF membranes were blocked with 3% BSA in 1 × TBST buffer (100 mM Tris–HCl, 150 mM NaCl, 0.5% Tween 20 at pH 7.5) at RT for 2 h. Membranes were then cut based on the molecular weight with reference to the protein ladder and incubated with the following primary antibodies (Proteintech) at 4 °C overnight: 1:5,000 mouse monoclonal anti-β-actin (clone: 7D2C10) and 1:2,000 rabbit polyclonal anti-PSA. After being washed with 1xTBST on a shaker for 30 min, 1:10,000 corresponding HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Proteintech) were added for a 1 h incubation at RT. Membranes were scanned with a Bio-Rad ChemiDoc™ Touch Imaging System with a chemiluminescence kit (Bio-Rad).

Multicolor imaging using an integrated smartphone prototype

A field-portable fluorescence imaging setup was enabled by a smartphone. The box module was digitally designed in SolidWorks software and printed via stereolithography with photosensitive curing resins by a UnionTech Lite 300 3D printer. The BP filter adaptors were cuboids with a length of 200 mm and a width of 33 mm to accommodate filters of all sizes. Two sets of band-pass (BP) filters (Shenzhen Yu Sheng Electronic Technology) consisted of 470 ± 10, 550 ± 10, 650 ± 10 nm for excitation, and 520 ± 10, 590 ± 10, and 680 ± 10 nm for emission, respectively. The filters were placed into 3D-printed inserts that situated the module. A 10 W integrated white-light LED was utilized to introduce BP-filtered illumination as the excitation source and set apart from the imaging window to create an incident oblique excitation and minimize excitation interference. The imaging window was set as a square with a side length of 15 mm. The power supply of LED was transformed by a 220 V AC to 12 V DC inverter to prevent undesirable stroboscopic effects. The height of the module was adjusted to 157 mm for observable focus planes on well plates. The length and width of the module were adjusted to 167 and 127 mm, respectively, to support most smartphones available in the market. BP interference filters for emission were installed in front of the camera to reject scattered and reflected excitation light and optimize fluorophore-specific emission.

Biotinylation of BSA

BSA was biotinylated at the reactive lysine side chains by adding 30-fold molar equivalence of sulfo-biotin-NHS to a BSA solution in pH 8 PBS. After 2 h incubation at RT, excessive biotin was removed by NAP purification columns. Next, the biotin labeling efficiency was quantified using 4′-hydroxyazobenzene-2-carboxylic acid (HABA). HABA binds SA to produce a colorimetric complex with strong absorption at 500 nm. Solvent-accessible biotin on BSA displaces bound HABA for its higher affinity with SA, causing absorption to decrease proportionately. The HABA-SA mixture of 0.5 mg mL−1 SA and 300 μM HABA was prepared in PBS. A fivefold dilution of biotin-BSA was added to the HABA-SA mixture and equilibrated for 30 s to record changes in OD500.

Covalent modification of capture antibody with SA

Primary amine groups of the anti-PSA capture antibody (cAb) were modified into sulfhydryl groups using Traut’s reagent (2-iminothiolane) at a molar ratio of 1:10 in pH 8 PBS (5 mM EDTA, DMSO < 1% v/v) for 30 min. The thiolated cAb was then purified by ultracentrifugation. In the meantime, amines of SA were modified into maleimides by sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) in pH 7.4 PBS at a molar ratio of 1:10. After 1 h of incubation, the activated SA was purified by ultracentrifugation and then mixed with the thiolated cAb in pH 7.4 PBS containing 5 mM EDTA in threefold molar excess. Finally, the reaction was rotated at RT for 1 h, and the SA-cAb complex was purified by an Amicon® MWCO 100 kDa ultracentrifuge filter (MilliporeSigma).

Enhanced fluorescence immunoassay on AgNIS

PS or AgNIS was immersed in 20 μg mL−1 biotin-BSA in PBS at 4 °C overnight. Wells were then washed three times with PBS and blocked with 3% BSA in PBS for 30 min, followed by 1 h incubation at RT in a 0.1% BSA PBS solution with 15 μg mL−1 SA-cAb. After washing three times with PBS, 50 μL PSA calibration standards at various concentrations in 0.1% BSA PBS were added for a 1 h duration of antibody-antigen interaction at RT. Immediately after washing, wells were incubated in 50 μL 0.1% BSA PBS containing 5 μg mL−1 CF647-labeled anti-PSA detection antibody (CF647-dAb) for 1 h at RT. At last, they were washed, dried, and scanned for fluorescence. The putative assay time starting from antigen addition was approximately 2 h. 10 μL samples were diluted to 50 μL with 0.1% BSA in PBS for serum measurement. Limit of detection (LOD) was calculated as 3 times of standard deviation (s.d.) of the blank with no antigens.

Conventional ELISA

High-bind 96-well PS microplates (Corning) were used for ELISA with the same antibody pair. The plate was coated with 50 µL 5 µg mL−1 cAb at 4 °C overnight, followed by blocking with 3% BSA in PBS for 1 h at RT. Identical PSA standards as in eFIA were applied for 1 h incubation at RT. Following plate washing, 2 µg mL−1 biotin-dAb in 0.1% BSA PBS was dispensed and incubated for 1 h. Subsequently, the plate was washed and incubated in a 1:2,000 dilution of SA-HRP (Beyotime) for 1 h. After washes, 100 µL 3,3′,5,5′-tetramethylbenzidine (TMB, Beyotime) substrate was added to each well for 30 min with reaction stopped by a half-volume 2 M H2SO4. Optical density (OD) was measured at 450 nm for quantification.

Detection of secreted PSA in PCa cell culture with AgNIS

The expression and shedding of PSA in PC-3 and LNCaP human PCa cell lines were studied by eFIA on AgNIS. First, 1 × 106 cells per well were seeded in a 6-well plate and cultured for 12 h in a 37 °C incubator. The culture media were then replaced with serum-free DMEM media containing DMSO, dihydrotestosterone (DHT, TargetMol), enzalutamide (ENZA, Shanghai Aladdin Biochemical Technology), or DHT plus ENZA (DMSO v/v < 0.5% in all cases to avoid cytotoxicity) for another 24 h incubation. At last, cells were harvested and lysed for expression analysis. Meanwhile, the culture media were also collected and centrifuged at 5,000 rpm for 10 min to remove any cell fragments, with supernatants subject to secretion analysis.

Detection of PSA in PCa tumors of human xenograft models

Inbred homozygous (Foxn1nu/Foxn1nu) mutant male BALB/c nude mice of 4–6 weeks were purchased from GemPharmatech and used for tumor inoculation in agreement with the animal protocol #L102012019120A approved by the Institutional Animal Care and Use Committees at Sun Yat-sen University Cancer Center (SYSUCC). Typically, 3–10 × 106 PCa cells in a 200 μL media:Matrigel (Corning) mixture (2:1 v/v) were injected subcutaneously near the right upper-flank. Xenograft mice bearing PC-3 and LNCaP tumors (n = 3) were sacrificed 30 and 20 days after grafting, respectively. Subcutaneous tumors were resected and placed in an Eppendorf tube containing 1 × RIPA lysis buffer with protease inhibitors and two ceramic grinding beads. Tissues were homogenized for 10 min at 70 Hz and -35 °C with a Luka Bead-mill Tissue Lyser (Guangzhou Luka Sequencing Instruments). Lysates were centrifuged at 12,000 g for 20 min with supernatant collected for analysis. Prostate glands (n = 3) were included as controls.

Microwave acceleration

Microwave acceleration was experimentalized on the interaction between AgNIS surface-immobilized biotin-BSA and CF647-labeled streptavidin (SA-CF647) under microwave irradiation. The microwave power and irradiation time were precisely controlled by an MCR-3E Lab Chemistry Microwave Reactor (Zhengzhou Yarong Instrument). The unmodified AgNIS surface without biotin-BSA was irradiated as a control for background subtraction. To best preserve native antigenicity of PSA in initial eFIA steps, the microwave was only applied to facilitate the binding of captured PSA and CF647-dAb. The instantaneous temperature elevation on the plasmonic surface in response to the microwave was measured in PBS with a FLIR ONE Pro thermal imaging camera.

Human serum

Approval for the human protocol #B2019-121-01 in this study was acquired from the SYSUCC ethics committee for investigational purposes. Sera from a cohort comprised of PCa patients (n = 50) and healthy subjects (n = 19) were collected on-site or from the Tumor Biobank at SYSUCC between 2019 May and 2020 Nov. All PCa patient samples were confirmed upon diagnosis by histopathological examination. Pre-interventional blood samples were drawn in venipuncture tubes on the day of interventions. In contrast, post-interventional blood was collected 4 days after RALRP or hormone therapy based on the TNM staging. After being centrifuged at 2,000 rpm for 10 min, serum and plasma were preserved from gel yellow-topped (clot activator and serum gel separator) and green-topped (sodium heparin) venipuncture tubes, aliquoted, and stored at -80 °C before use. Anticoagulated whole blood was kept at 4 °C and tested within 7 days following collection. Clinical PSA levels were measured with an Elecsys® total PSA kit on the Roche cobas e 602 analyzer by ECLIA.

Statistical analysis

Statistical analysis was performed using Microsoft Excel and SPSS Statistics. Data were presented as mean ± s.d. denoted by error bars from a minimum of three replicates unless otherwise noted. Statistical significance was assessed by two-tailed paired or unpaired Student’s t-tests. Neither blinding nor randomization was performed on the subjects. No prior statistical analysis was used to predetermine the sample size for enrollment.

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