shell nanoparticles with plasmonic simulation insights

Sensors & Actuators: B. Chemical 299 (2019) 126956

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Multiplexed surface plasmon imaging of serum biomolecules: Fe3O4@Au Core/shell nanoparticles with plasmonic simulation insights

T

Gayan Premaratnea, Asantha C. Dharmaratnea, Zainab H. Al Mubaraka, ⁎ Farshid Mohammadparastb, Marimuthu Andiappanb, Sadagopan Krishnana, a b

Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078, United States School of Chemical Engineering, Oklahoma State University, Stillwater, Oklahoma 74078, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Multiplex imaging serum proteins serum miRNAs wide dynamic range FDTD simulation core/shell nanoparticles

Nano-biosensors that are not only sensitive and selective, but also enable multiplex detection of ultra-low levels of both large and small biomolecules in clinical sample matrices are essential for in vitro diagnostics. We present herein a multiplex surface plasmon microarray design that employs citrate-stabilized Fe3O4@Au core/shell nanoparticles (NPs) as the plasmon signal amplification label for combined detection of serum proteins and nucleotide markers. The multiplex sensing is demonstrated using two interleukins (IL-6 and IL-8) and two microRNAs (miRNA-21 and miRNA-155) in 10% serum, which is clinically relevant than simple buffer solution based biosensors. We observed that the surface plasmon signal change for larger proteins even at higher concentrations was less than the relatively smaller miRNA molecules. We draw two conclusions from this result: (i) the number of selectively bound analytes onto the sensor (i.e., antigen for an antibody or miRNA for a capture nucleotide) influences the signal change, and (ii) the extent of interaction of the detection probe carrying core/ shell NP labels with the sensor surface plasmons influences the amount of signal change. Results indicate that both factors, (i) and (ii), are greater for small oligonucleotide hybridization assembly than a large sandwich protein immunoassembly. The core/shell NPs offered several fold enhanced sensitivity and wider dynamic range of detection over assays without using them. With recently growing attention on in vitro diagnostics for painless/ minimally-invasive detection of diseases and abnormalities, findings presented herein are important for designing novel multiplex biosensors for real sample analysis in complex matrices.

1. Introduction Circulating biomarkers have been recognized as important target molecules not only for diagnoses of cancer and other deadly disorders but also for monitoring treatment outcomes and post-surgery recurrence. To increase prediction rates and reduce false positive diagnoses, it is important to measure a panel of key biomarkers than conventional analysis of a single marker. Highly expressed circulating protein and microRNA (miRNA) markers have received considerable attention in liquid biopsy studies due to their promising predictability feature [1]. Many analytical methods, including enzyme-linked immunosorbent assay, spectroscopic, electrochemical and molecular biology techniques (real-time polymerase chain reaction, northern blotting, microarray technology, and in situ hybridization), have been employed in cancer biomarker detection [2–7]. In view of developing molecular technologies that enable more precise and objective decision making, surface plasmon resonance

⁎

(SPR) spectroscopy with multiplexed imaging is an attractive strategy. SPR is a highly sensitive surface analysis technique that can measure the binding events of various ligands to their respective receptors through refractive index changes [8,9]. Additionally, SPR can be used for high-throughput screening and to obtain real-time binding insights about biomolecular interactions by tuning the surface chemistries and metal-dielectric interfacial properties [10–12]. Due to these features, SPR methods have been widely employed for selective detection of various biomarkers [13–15]. Incorporation of nanomaterials in bioassays offers the unique advantages of robustness from superior chemical and solvent stability compared to light-sensitive labels, versatility of use in various analytical techniques, increased sensitivity and lower detection limits (DLs). However, the bottleneck of poor dynamic range of detection needs to be overcome to develop nanomaterial-based assays for measuring widely spanning analyte concentrations in clinical samples [16,17]. Among metal nanoparticles (NPs), bimetallic NPs allow tuning of

Corresponding author. E-mail address: [email protected] (S. Krishnan).

https://doi.org/10.1016/j.snb.2019.126956 Received 26 June 2019; Received in revised form 5 August 2019; Accepted 6 August 2019 Available online 10 August 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

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(sequences presented in Table S1), bovine serum albumin (BSA), dithiothreitol (DTT), 6-mercaptohexanol (MHOH), 3-mercaptopropionic acid (MPA), ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS), gold(III) chloride trihydrate (HAuCl4.3H2O), sodium borohydride, sodium citrate, gold and iron standards for ICP analysis (1000 ppm), and gold nanoparticles (of similar average hydrodynamic diameter as the core/shell NPs) were obtained from Sigma-Aldrich (St. Louis, MO). Recombinant IL-6 and IL8 proteins and purified anti-human IL-6 and IL-8 sandwich antibodies (the surface-immobilized antibody is monoclonal and the core/shell NPs attached detection antibody is polyclonal) were purchased from BioLegend Inc. (San Diego, CA). The human serum was purchased from Fitzgerald Industries International Inc. (North Acton, MA). Citrate-stabilized magnetic nanoparticles (Fe3O4 NPs, 50 nm hydrodynamic diameter) were purchased from Chemicell GmbH Inc. (Berlin, Germany). Ultrapure deionized water (DI water) and NAP-10 columns were obtained from GE healthcare (Cranbury, NJ). SpotReady-16 gold spotted glass microarray chips (spot size 1 mm diameter, SPR-1000-016) were obtained from GWC Technologies (Madison, WI).

plasmonic and magnetic properties, and the core (inner component)/ shell (outer component) NPs is an important class because its members combine the beneficial properties of the two different nanomaterials [18,19]. Different bimetallic combinations, such as silver@gold, iron@ platinum, platinum@cobalt, and gold@platinum, have been designed for biosensing, bioimaging, and drug delivery applications [20]. In particular, the magnetic properties of iron-gold (Fe3O4@Au) bimetallic core/shell NPs offer the dual benefit of (i) easy conjugation and magnetic separation of desired compounds for subsequent detection in the assay [21], and (ii) the plasmonic gold shell properties are useful for amplifying the surface plasmon detection signals [22]. In addition, coating of Fe3O4 NPs with gold provides more dispersibility and room for various surface functionalization strategies (e.g., thiols, polymers, small molecules, and dendrimers) for immobilizing biomolecules [23,24]. Brown et al. demonstrated the synthesis of homogeneous and large NPs via a seeding method involving the reduction of gold salts using either sodium citrate or hydroxylamine reagents [25]. Pham et al. magnetically separated IgG protein using gold-coated Fe3O4 NPs of 15–40 nm size [21]. Prior studies reported the application of Fe3O4@Au NPs and its derivatives for electrochemical [26], chemiluminescent [27], and surface enhanced Raman scattering [28], based assays for detection of various biomolecules. And a conventional SPR immunosensor based on a Fe3O4@Au composite utilized protein detection in simple buffer solutions [29], which do not reflect the complexity/design challenges associated with real sample multiplex imaging analysis such as in serum. Moreover, the designed multiplex platform for combined detection of two proteins and two miRNA markers is novel and an important state-of-the-art diagnostic feature. Moreover, our multiplex sensor does not require any composite preparation materials and steps, hence it is relatively simpler in design, implementation, and analysis. Specifically, in this study, we devised a multiplexed SPR imaging (SPRi) microarray design utilizing Fe3O4@Au NPs to simultaneously measure two proteins and two miRNAs present in human serum. A greater plasmon enhancement signal in the imager by the synthesized Fe3O4@Au NPs over either Fe3O4 or Au NPs of similar hydrodynamic sizes are systematically compared in combination with Finite-difference time-domain (FDTD) simulation analysis. To demonstrate the applicability of our SPRi core/shell NP bioassay strategy for health monitoring based on circulating biomarkers, we chose interleukin-6 (IL-6), IL-8, miRNA-21, and miRNA-155 as representative biomolecules. IL-6 is one of the major cytokines found in the tumor microenvironment and in the circulatory system. It is over-expressed in all types of tumors, thereby depicting the progression and severity of disease [30]. The serum concentration of IL-8 correlates with tumor burden, thus making it a useful pharmacodynamics biomarker to detect responses to cancer immunotherapy [31]. Among the members of miRNA family, miRNA-21 is one of the consistently upregulated circulatory biomarkers detected in cancer patients [32]. Similarly, miRNA-155 is a robust oncogenic circulating miRNA, and its overexpression can indicate the promotion of tumorigenesis [33]. The circulating concentrations of miRNA-21 and miRNA-155 markers have the potential to be used for early diagnosis and monitoring of tumor development as well as for predicting chemoresistance. The analytical advantages of the designed sensor array strategy are in the detection of four biomarkers and the associated binding strength estimation in a serum matrix, which is vital to understand the degree of interaction of the chosen receptors with their target analytes. This SPRi multiplex sensing platform is useful for analysis of any biomarkers present in real clinical samples.

2.2. Instrumentation and techniques SPRimager-II array instrument (Horizon SPR imager model) operating at a SPR source wavelength of 800 nm at room temperature was used (GWC Technologies, Madison, WI, USA). The experiments were conducted using our custom designed four-channel microfluidics set-up each connected to a syringe pump (100-μL sample loop, New Era Pump System, Inc., NY, USA). The SPRi pixel intensities for increasing miRNA or IL concentration upon binding to their surface-immobilized selective receptors followed by the signal amplification from the binding of Fe3O4@Au NPs linked detection probes were measured. Digital Optics V++ software package provided with the instrument was used to collect the SPRi difference images (i.e., differences in the pixel intensities before and after the binding events), and the 3D-images were represented using ImageJ 1.49v software (NIH, USA). Quantification of oligonucleotides was done by a Nanodrop ND1000 spectrophotometer (Thermo Scientific, Waltham, MA). The UV–vis spectral absorbance of antibodies and DNA oligonucleotides was measured at 280 and 260 nm, respectively [34]. The elemental analysis of the Fe3O4@Au NPs was performed by an inductively coupled plasma optical emission spectrophotometric analyzer (ICP-OES, SPECTRO Analytical Instruments Inc., NJ, USA). The emission line selected for the Fe was 259.9 nm, and for Au, the line was 267.5 nm [35]. The hydrodynamic diameter and surface charges of only Fe3O4, only Au NPs, and that of Fe3O4@Au core/shell NPs and their covalently attached bioconjugates with detection antibody or DNA were determined using a ZetaPALS potential analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA). Five measurements were made at a 90° angle (2 min per measurement). The samples were diluted five times in phosphate buffered saline (PBS) prior to the measurements. The ζ-potential was calculated by phase analysis of light scattering (PALS), which determined the electrophoretic mobility of the particles by Smoluchowski's equation (available with the instrument software). Transmission electron microscopy (TEM, JEOL JEM-2100) images of the synthesized Fe3O4@Au NPs, starting 50 nm Fe3O4 NPs, and commercially purchased Au NPs of hydrodynamic diameter similar to the core/shell NPs were obtained by preparing drop-coated samples on carbon surface grids. Surface characterization of the gold microarray before and after coating with the surface capture DNA, after hybridizing with the miRNA marker, and subsequently with the plasmon enhancing Fe3O4@Au NPs linked detection probes were conducted by scanning electron microscopy (SEM, Model: FEI Quanta 600FE). An accelerating voltage of 20 kV was applied. The images were acquired using FEIxT Microscope Control Software (Fig. S2). FDTD simulations were used to understand the SPRi responses of Fe3O4, Au, and Fe3O4@Au NPs. The Lumerical FDTD package from

2. Experimental section 2.1. Reagents and materials Thiol modified custom-designed DNA oligonucleotides and miRNAs 2

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Scheme 1. Schematic illustration of the synthesis of Fe3O4@Au core/shell NPs using the reaction mixture consisting of 1. HAuCl4.3H2O, 2. Fe3O4 NPs, and 3. Sodium citrate.

hydrodynamic size of antibodies (10–15 nm) [40]. A similar size increase was observed upon chemisorption of thiol-activated DNA to the Fe3O4@Au NPs. The polydispersity index was ≤ 0.2, indicating good dispersion and narrow size distributions of the conjugated NPs in the buffer medium, which is likely the result of surface charge repulsions. The negative ζ-potential measured for the unconjugated core/shell NPs was due to the citrate surface groups. The anti-IL antibodies are members of an IgG class, which typically have an isoelectric point (pI) of 8–9.5 [41] and hence a net positive charge at pH 7.4. This is in agreement with the significant shift of the ζ-potential in the positive direction for the detection antibody attached Fe3O4@Au NPs. In contrast, the intrinsic negative charges of nucleotides [42] from their phosphate groups shifted the ζ-potential of DNA-Fe3O4@Au NPs to more negative values (Table 2). Together, the Zeta potential and hydrodynamic diameters are excellent diagnostic tools for the confirmation of synthesized core/shell NPs and their bioconjugates.

Lumerical Inc. was utilized to simulate the SPR scattering responses of the NPs. The empirical optical constants for Au and Fe3O4 were taken from the literature [36,37]. The computational domain was the physical region over which the simulation box was gridded inside and outside of the NPs. The mesh size used for the simulations was 0.5 nm. A Gaussian broadband pulse was used as the incident light source in the simulated wavelength region. The SPR scattering responses of the incident light source by the NPs over a wide range of wavelengths were obtained with a single simulation using the total-field/scattered-field (TFSF) formalism. The box sizes used for the simulation box, scatteredfield monitor, TFSF source, total-field monitor were 200, 80, 50 and 40 nm, respectively. The TFSF source was set to originate from the + zdirection. The boundary conditions used for the simulations were periodic in the x,y-directions and a perfectly matched layer in the z-direction. Detailed Experimental Procedures for the Synthesis of Fe3O4@Au core/shell nanoparticles (Scheme 1), preparation of the core/shell NPs conjugated detection molecules and preparation of the four-channel SPR microarray for multiplexed analysis (Fig. 1) are presented in the Supporting Information. From our present observation, the synthesized nanoparticles can be stored for at least 6 months up to a year at 4 °C with no changes on the hydrodynamic diameters and zeta potentials.

3.2. Estimation of SPRi signals of Fe3O4@Au NPs over similar hydrodynamic diameters of Fe3O4 or Au NPs alone To measure the SPRi signals, pixel intensities of only Fe3O4 NPs (100 nm hydrodynamic diameter, Chemicell Inc.), only Au NPs (100 nm hydrodynamic diameter, Sigma-Aldrich), and the synthesized Fe3O4@Au NPs (75 nm hydrodynamic diameter) or Fe3O4@Au NPs (105 nm hydrodynamic diameter) were compared in a manner similar to that reported previously [43]. In brief, the SPRi microarray was coated with a polycationic layer, polyethyleneimine (PEI, 0.1 mg mL–1 in DI water), by adsorption for 30 min followed by washing with DI water. The negatively charged citrate-stabilized NPs then were adsorbed for 30 min on the polymer-coated spots and rinsed with DI water to remove any unbound NPs. The difference images obtained (before and after the coating with NPs) are represented in a 3D format (ImageJ software, Fig. 2). The average pixel intensities for the Fe3O4@Au NPs (75 nm) were 6fold and 2.5-fold higher than Fe3O4 (100 nm) and Au NPs (100 nm) alone, respectively. The increase in the Au shell size in the case of 105 nm Fe3O4@Au NPs further increased the SPRi pixel intensity to ˜7fold and 3-fold over the Fe3O4 (100 nm) and Au NPs (100 nm) alone, respectively. Hence, for subsequent studies, for biosensing of the protein and miRNA markers, we used the 105 nm sized Fe3O4@Au NPs. Results obtained infer that although SPR signal amplifications based on either magnetic or gold NPs have been reported, the combination of these two NP systems as a bimetallic core/shell type offers significantly greater signals than the individual NPs of similar hydrodynamic

3. Results and discussion 3.1. Hydrodynamic size and Zeta (ζ)-potentials of only Au NPs, only Fe3O4 NPs, Fe3O4@Au core/shell NPs and their conjugates The average hydrodynamic diameters and Zeta potentials of the synthesized Fe3O4@Au core/shell NPs and the individual NPs (citratestabilized 50 nm Fe3O4 NPs starting material and Au NPs of similar hydrodynamic diameter as the core/shell NPs) are presented in Table 1. The negative surface charge of all NPs is evident from the negative Zeta potential values. More importantly, the formation of core/shell NPs shifted the Zeta potential to more negative values than either of the individual NP types. Table 2 presents the hydrodynamic diameters and Zeta potentials of Fe3O4@Au core/shell NPs and their conjugates with the detection molecules (second IL-6 antibody or oligonucleotide). The antibodies contain free surface lysine residues to covalently attach them to the surface carboxyl groups on Fe3O4@Au NPs at random orientations, and no additional steps were implemented for orienting the antibodies. The average size of the Fe3O4@Au NPs increased by about 25 nm after antibody attachment. This is consistent with the typical 3

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Fig. 1. A. The experimental set-up for multiplexed SPRi analysis using our in-house designed four-channel flow injection system. B. The SPRi chip was modified with capture probes (CAb: capture antibodies or CDNA: capture DNA) and the analytes were assayed as follows: Two lanes (4 spots each) of the SPRi microarray were selfassembled with a monolayer of MPA. The remaining two lanes were self-assembled with thiol-activated hairpin capture DNAs of miRNA-21 and miRNA-155 (4 spots each) followed by blocking the free surface with MHOH. The IL-6 and IL-8 capture antibodies were covalently attached to the −COOH activated MPA surface [38,39] (four spots each) followed by blocking of the free surface with 1% BSA. To develop the method, various concentrations of the protein and miRNA markers were spiked in 10% human serum and allowed to bind their respective capture molecules on the chip using the designated individual flow channels. The signal amplification step of the bioassay was subsequently followed up by introducing the respective detection molecules (DAb: detection antibodies or DDNA: detection DNA) conjugated to Fe3O4@Au NPs.

100 nm diameter, and core@shell Fe3O4@Au NPs of 10 nm core size and varying shell thicknesses. For comparison, the normalized scattering spectra of Fe3O4 NP of 10 nm diameter is also shown in Fig. 3. The size range of the NPs used in the simulations for the results shown in Fig. 3 is based on the average sizes of the NPs calculated from the TEM images of the respective NPs (Figs. S1 – A to C). As seen in Fig. 3, Fe3O4 NPs exhibit no significant localized surface plasmon resonance (LSPR) scattering peak in 400–1200 nm region. The Au nanosphere of 100 nm diameter shows LSPR scattering peak centered at 550 nm. On the other hand, Au nano-shell in Fe3O4@Au NP exhibits multiple LSPR scattering peaks in the 600–1000 nm region. These peaks are expected to be a result of LSPR dipole and higher order modes in Au nano-shell. Thus, the FDTD-simulation results in Fig. 3 shed light on the presence of LSPR scattering peaks of Au nano-shell that resonate with the source wavelength of our instrument (800 nm). The FDTD simulation results suggest that, for an 800 nm incident light

Table 1 Characterization of the synthesized core/shell Fe3O4@Au NPs shown with starting 50 nm Fe3O4 NPs and comparison to only Au NPs of similar hydrodynamic diameter as the core/shell material.

Fe3O4 NPs Fe3O4@Au NPs Au NPs

Hydrodynamic diameter (nm)

Polydispersity

ζ potential (mV)

50 ± 6 105 ± 6 106 ± 4

0.24 ± 0.01 0.19 ± 0.03 0.06 ± 0.01

−10.5 ± 0.6 −38 ± 4 −19.2 ± 1.6

diameters.

3.3. FDTD simulations Fig. 3 shows the normalized SPR scattering spectra of Au NP of

Table 2 Hydrodynamic diameters and ζ-potentials of Fe3O4@Au NPs and their conjugates with detection antibody (shown here for IL-6 antibody) or the detection DNA probe. Parameter

Fe3O4@Au NPs

Antibody-Fe3O4@Au NPs

DNA-Fe3O4@Au NPs

Hydrodynamic diameter (nm) Polydispersity index ζ-potential (mV)

105 ± 6 0.19 ± 0.03 –38 ± 4

132 ± 11 0.18 ± 0.05 –12 ± 1

124 ± 5 0.15 ± 0.01 –51 ± 3

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Fig. 2. The SPRi responses of A. 100 nm Fe3O4 NPs (20 ± 3 pixels), B. 75 nm Fe3O4@Au NPs (119 ± 6 pixels), C. 100 nm Au NPs (48 ± 5 pixels), and D. 105 nm Fe3O4@Au NPs (152 ± 4 pixels) adsorbed onto a PEI layer modified gold surface (N = 3). (a) Schematic and experimental 3D images of SPR pixel intensities and (b) only the PEI layer modified gold surface with no NPs. The corresponding line profiles and raw difference images (in grey) are shown on the right.

3.4. Quantitation of capture and detection molecules used in the designed SPRi microarray The fabricated SPRi microarray includes a sandwich immunoassay for IL detection and a dual hybridization strategy for assaying miRNA molecules present in a serum matrix and signal amplification with Fe3O4@Au NPs carrying detection probes. Table S2 presents the results of spectrophotometric quantification of the amounts of capture DNA and capture antibodies immobilized on the microarray surface, and the amounts of detection probes attached to Fe3O4@Au NPs. This quantitation confirmed the successful immobilization of the biomolecules on the desired surfaces. 3.5. Real-time analysis of serum biomarkers Fig. 4 presents the real-time sensograms, raw difference images, 3-D representations, and line profiles corresponding to the multiplexed detection of ILs and miRNAs spiked in 10% human serum. Additional real-time sensograms showing SPRi responses for different concentrations of the target biomarkers are presented in Figure S3. The signals were amplified several folds by the introduction of the bimetallic NPdetection probe step following the analyte marker binding. The observed Fe3O4@Au NP induced signal amplification was proportional to the serum biomarker concentration, which further confirmed the selective interaction of the detection molecules with the target biomarker bound microarray surface capture probes. Interestingly, Fig. 4 shows that the surface plasmon detection signal for the larger sized proteins even at a several fold higher concentration was lower than the relatively smaller miRNAs, which led to the following conclusions: (i) the significance of the ratio between the bound protein or miRNA and their surface immobilized probe molecules is an important factor on the magnitude of signal output, and (ii) the extent of plasmonic interactions between core/shell NPs and surface gold plasmons is greater for a shorter hybridization assembly bridge compared to a larger sandwich antibody-antigen-antibody immunocomplex. Fig. 5 data plot shows the SPRi response plots for increasing biomarker concentration that was subtracted for unspiked control serum signal. Also shown is the sensitivity enhancement offered by the Fe3O4@Au NPs attached detection probes. Sensitivity enhancements of

Fig. 3. Simulated SPR scattering spectra of spherical Au NP of 100 nm diameter and core@shell Fe3O4@Au NPs of 10 nm core size and varying shell thicknesses (1–3 nm) as labeled in the plot. Also shown is the simulated scattering spectra of Fe3O4 NP of 10 nm diameter showing no peaks.

excitation, when the Au nanoshells (Au spherical or that in core@shell Fe3O4@Au NPs) are placed near the surface of a gold film, the plasmonic coupling can happen between the SPR mode of Au film and the localized LSPR mode of the surrounding core-shell nanoparticles. This coupling can favor higher reflectivity change for a given angle of incidence, which is predominant in the core@shell NPs due to their resonating wavelength with the incident light in our system. Furthermore, Fig. 3 shows that the SPR peak of Fe3O4-Au nanoshell blue-shifted with increasing Au shell thickness. With increasing shell thickness, the geometry of Au nanoshell approaches the geometry of a gold nanosphere and hence the blue shift is expected. This possibility is consistent with the previously reported experimental observations for Au nanoshells [44,45].

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Fig. 4. SPRi responses for the multiplexed assay on the designed 4-channel flow injection analysis. A. Real-time pixel intensity changes for 10% serum spiked with a. IL-8 (10 nM), b. IL-6 (10 nM), c. miRNA-21 (250 fM), and d. miRNA-155 (250 fM). I. Represents the introduction of 10% serum spiked with the markers to the capture probe immobilized microarray and II. Represents the introduction of the detection probes conjugated to Fe3O4@Au NPs. B. Final difference image of the test spots after detection of protein and miRNA markers in a single microarray, C. 3-D representation and D. SPR intensity line profile for a row of four spots in the array.

about 3-times for the ILs and about 7-times for the miRNAs were achieved by the Fe3O4@Au NP bioconjugation strategy. In addition to the signal enhancements, the dynamic ranges widened by the introduction of the detection molecules conjugated Fe3O4@Au NPs (Table 3). The observed differences in the dynamic ranges among proteins and miRNAs are attributed to the several orders of magnitude greater miRNA capture probes (due to smaller size and self-assembled monolayer arrangement accommodating a large number of molecules) immobilized on the surface over the large antibody molecules (Table S2). This in turn can offer binding sites for a wider concentration range of miRNA markers and detection in a linear manner. In contrast, steric effects and random antibody orientations from carbodiimide coupling chemistry could limit the antigen access to the nearest neighboring

Table 3 Dynamic ranges observed for the tested biomolecules spiked in 10% serum with and without the use of Fe3O4@Au NPs (Fig. 5). Target molecule

Without Fe3O4@Au NPs

With Fe3O4@Au NPs

miRNA-21 miRNA-155 IL-6 IL-8

100 – 500 fM 50 – 500 fM 40 pM – 2 nM 20 pM – 2 nM

50 fM – 2 pM 25 fM – 4 pM 10 pM – 100 nM 8 pM – 75 nM

antibody sites on the surface and a narrower saturating concentration signal. The DLs for miRNAs and ILs were calculated by the following equation: DL = [3 x (standard deviation of the control/slope of the

Fig. 5. SPR response plots for protein markers (IL-6 and IL-8) and miRNA markers (miRNA-21 and miRNA-155) in 10% human serum: a. after and b. before introducing the signal amplifying detection probes conjugated to Fe3O4@Au NPs. (N = 3 replicates). 6

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Fig. 6. A. Changes in SPRi intensities upon the binding of Mixture a [IL-8 (5 nM), miRNA-21 (250 fM), and miRNA-155 (250 fM) spiked in 10% serum] or IL-6 alone (5 nM) spiked in 10% serum to I. IL-6 capture antibody modified surface. Similarly, Mixture b [IL-6 (5 nM), IL-8 (5 nM), and miRNA-155 (250 fM) mixture spiked in 10% serum] or miRNA-21 250 fM alone spiked in 10% serum upon introduction to the miRNA-21 capture DNA modified surface. II. SPR response for a mixture containing the detection probes conjugated to Fe3O4@Au NPs. B. Cross-reactivity for the mixture of non-specific molecules spiked in 10% serum relative to the specific marker responses, IL-6 and miRNA-21 (N = 3 replicates).

responses in Fig. 6-B shows that the mixtures A and B show only 5% and 9% cross-reactivity with the capture molecules of IL-6 and miRNA-21, respectively. These results confirm the specificity of the capture molecules immobilized on the multiplexed array surface toward selectively recognizing their related specific target serum biomarker.

regression line)]. The DLs for IL-6, IL-8, miRNA-21, and miRNA-155 were 28 pM, 18 pM, 502 fM, and 483 fM, respectively. These DLs and dynamic ranges were compared to those of other recent SPR assays that detected protein or miRNA biomarkers in various matrices using different amplification strategies (Table S3). Our methodology is unique in quantifying the serum protein and miRNA biomarkers by a multiplexed detection format. The higher DLs when measuring a marker in a serum medium over a simple buffer solution can infer the undesirable effect of complex serum sample matrix (Table S3 comparison).

4. Conclusions We demonstrated here a multiplexed surface plasmon microarray exploring the favorable plasmon enhancing properties of iron oxide/ gold core/shell nanomaterials. The combined magnetic and plasmon enhancing features of the core/shell nanomaterial were useful for magnetically separating the attached detection probes easily and amplifying the SPRi signal output while minimizing the non-specific signals arising from the serum matrix. These features improved the selectivity, sensitivity, and dynamic range of the assay. FDTD simulations inferred the characteristics scattering peaks of the core/shell NP causing increased SPR signal intensity with a matching resonance wavelength of the source light. The estimated binding constants in the μM to nM range provide evidence for strong affinities between the analyte biomarkers and their receptor molecules on the designed sensor array surface. Broader applications of this approach to other proteins and miRNA biomarkers present in serum or other similar complex biological matrices is anticipated. Our future direction is to further lower the detection limits and enable a tunable dynamic range of detection by expanding the demonstrated detection strategy.

3.6. Analysis of binding strength of protein and miRNA markers Determining binding kinetics is important for assessing the strength of interactions between the markers and their receptors on the designed array surface. Additionally, the binding constants can be used as a quality control parameter to reproducibly make and use the bioassays for reliable large-scale applications and promptly diagnose when deviations occur from the optimized performance. The Langmuir adsorption isotherm was employed to determine the equilibrium dissociation kinetics based on a 1:1 bimolecular interaction model as detailed in the Supporting Information [46]. Figure S4 displays the plots of relative surface coverage (θ) versus the concentration of each biomarker. The data points were fit into the Langmuir isotherm (eq. S-2) using OriginPro software (Originlab, Northampton, MA). The differences in the shapes of the Langmuir isotherm fits for ILs and miRNAs denote the corresponding variations in the association rates [47]. The Kads values obtained for IL-6, IL-8, miRNA-21, and miRNA-55 were 2.50 ( ± 0.17) x 107 M–1, 6.10 ( ± 1.08) x 107 M–1, 7.00 ( ± 0.76) x 108 M–1, and 7.47 ( ± 1.10) x 108 M–1, respectively, which agree with the signal intensity data. At a bulk concentration equal to 1/Kads, the concentration of the capture probe corresponds to 50% surface coverage (Cθ0.5). We determined the Cθ0.5 values of 40.1, 22.4, 1.4, and 1.2 nM for IL-6, IL-8, miRNA-21, and miRNA-155, respectively.

Acknowledgments We thank the College of Arts & Sciences, Oklahoma State University for financial support. M. A. acknowledges funding support from the Oklahoma Center for Advancement of Science and Technology (Award # HR18-093). Appendix A. Supplementary data

3.7. Assay cross-reactivity assessment

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.126956.

Cross-reactivity is an important parameter to evaluate for any analytical bioassays. This is particularly significant when the analysis is performed in a complex sample matrix such as serum, which contains several other non-specific biomolecules. To address the assay cross-reactivity for other measured analytes in the serum, two mixtures [Mixture a: IL-8 (5 nM), miRNA-21 (5 pM), and miRNA-155 (5 pM); and Mixture b: IL-6 (5 nM), IL-8 (5 nM), and miRNA-155 (5 pM)] in 10% serum were introduced onto an array surface that contained the IL-6 capture antibody or the capture-DNA of miRNA-21 (Fig. 6-A, step I). Additionally, the signal amplification step with the detection probes attached Fe3O4@Au NPs was followed up (Fig. 6-A, step II). The SPRi

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