Ultrasensitive Detection of Cancer Prognostic miRNA Biomarkers

Article pubs.acs.org/acssensors

Ultrasensitive Detection of Cancer Prognostic miRNA Biomarkers Based on Surface Plasmon Enhanced Light Scattering Chih-Tsung Yang,† Mohammad Pourhassan-Moghaddam,‡ Lin Wu,§ Ping Bai,§ and Benjamin Thierry*,†,∥ †

Future Industries Institute and ∥ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Mawson Lakes Campus, Mawson Lakes, South Australia 5095, Australia ‡ Department of Medical Biotechnology and Immunology Research Center, Tabriz University of Medical Sciences, Tabriz 51368, Iran § Electronics and Photonics Department, Institute of High Performance Computing, Agency for Science, Technology, and Research (A*STAR), Singapore 138632 S Supporting Information *

ABSTRACT: The development of simple yet ultrasensitive biosensing approaches for the detection of cancer prognostic microRNA is an important step toward their successful clinical implementation. We demonstrate the relevance for the detection of circulating miRNA of a novel signal amplification scheme based on surface plasmon resonance enhanced light scattering (SP-LS). In addition to experimental optimization carried out using gold nanoparticle (AuNP) tags conjugated with a monoclonal antibody with high affinity for RNA*DNA hybrid duplexes, simulation modeling was conducted to obtain insights about SP-LS biosensing. SP-LS enabled the detection of miRNA122 at subpicomolar concentrations within 30 min, and a limit of detection of 2 attomoles (60 fM, 50 μL) was determined. MiRNA-122 could also be reliably detected in a high concentration background of nontarget miRNA. The proposed SP-LS miRNA detection approach could be readily applied to other miRNA targets of diagnostic importance and further developed to allow for multiplex measurements of miRNA panels. The promising results obtained in this study and advantageous features of SP-LS warrant further development and its application to clinical samples. KEYWORDS: surface plasmon resonance, surface-plasmon-enhanced light scattering, cancer prognostic, miRNA, gold nanoparticles, amplification

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high throughput format and eliminate some of the issues associated with qRT-PCR.6 However, despite the exquisite sensitivities of sequencing based detection methodologies, their requirement for costly equipment and tedious process of postsequencing data analysis limit their clinical applicability. Overall, considering the challenging features of miRNA biomarkers, namely, their small size in sequence, easy degradation, and low abundance, it is indispensable to develop technologies able to meet the requirements for fast, sensitive, and accurate detection of target miRNAs. To meet these challenges, a number of solid state biosensing technologies are being developed for the specific and sensitive detection of miRNA in biological fluids.7−10 Surface plasmon resonance (SPR) is widely used in the field of molecular biosensing. However, owing the inherent low molecular weight of miRNA, signal amplification is typically required to achieve reliable detection at clinically relevant concentrations with SPR. The pioneering work from Corn’s group demonstrated the tremendous potential of AuNP amplification schemes for the

icroRNAs (miRNAs) are small non-protein-coding RNA molecules that regulate gene expression by binding to complementary sequences in target mRNAs and mediating translational repression and/or mRNA degradation.1 MiRNAs are frequently dysregulated in many diseases including neurodegenerative diseases, cancer, and cardiovascular diseases. There is therefore a significant interest in their potential as biomarkers, especially since it has been established that miRNAs exist in highly stable forms in human biological fluids.2 A number of miRNA cancer diagnostic and prognostic biomarker panels have already been validated.3,4 For example, a miRNA panel associated with breast cancer could provide accurate prediction of tumor relapse and survival in patients.5 Hence, the rapidly growing field of miRNA biology demands novel and robust methods for their accurate and sensitive detection and quantification. The current gold standard methods for the detection of miRNAs in biological fluids rely on PCR-based assays, including quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). However, PCR amplification of miRNA is not trivial which adds to the inherent complexity and cost of qRT-PCR assays. Next generation sequencing allows for simultaneous sequencing and quantification of miRNAs in a © 2017 American Chemical Society

Received: December 1, 2016 Accepted: April 13, 2017 Published: April 13, 2017 635

DOI: 10.1021/acssensors.6b00776 ACS Sens. 2017, 2, 635−640

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hybrid duplexes were recognized with the mouse monoclonal antibody (D5H6) supplied by Covalab. Long chain PEG (HS-PEG-OMe; MW = 2000 Da) was purchased from Rapp Polymere and short chain PEG (HS-PEG-COOH; MW = 458.6 Da) was acquired from Polypure. Preparation and Modification of SPR Sensor Chips. SPR sensor chips were sputter-coated with 48 nm of gold film on top of a thin 2 nm of Cr layer. The chips were incubated with the capture thiolated DNA probe in buffer (4 μM, PBS) overnight. After a short rinse with PBS, the chips were passivated with MCH (10 μM, PBS) for 30 min. The sensor surface was then washed with PBS. Preparation and Bioconjugation of AuNP Tags. The 55 nm AuNPs were synthesized using the citrate reduction method as previously reported.16 The as-synthesized AuNPs were PEGylated with a mixture of long PEG (HS-PEG-OMe; MW = 2000) and short PEG (HS-PEG-COOH; MW = 458.6) using a molar ratio of 1 to 2 in Milli-Q water overnight. The PEGylated AuNP solution was centrifuged at 6000 rpm for 10 min and washed with milli-Q water twice to remove unbound PEG. The AuNPs pellet was collected in the Eppendorf tube at the final volume of 150 μL (optical density (OD) = 40). The terminal carboxylate groups were then activated with 20 μL of EDC (0.4 M) and NHS (0.1 M) for 10 min. After centrifugation at 6000 rpm for 10 min, the supernatant was removed and the pellet was redispersed with 80 μL of PBS buffer. 20 μL of antibody (1 mg/mL) was added into the tube and incubated for 3 h at rt. After centrifugation at 4 °C and 6000 rpm for 10 min, the supernatant was removed and the pellet was redispersed in 500 μL of PBS. The unreacted ester groups were deactivated with 20 μL of ethylamine (1 M, pH = 8.5) for 5 min which was followed by washing with PBST buffer (PBS + 0.05% Tween) twice. The antibody modified AuNPs were then blocked with 1% BSA in PBST buffer and kept at 4 °C until used. The successful synthesis and bioconjugation of AuNPs was monitored by UV−vis (Figure S1a), DLS (55.3 ± 0.7 nm, PDI = 0.06 ± 0.01, Figure S1b), and SEM (Figure S1c). miRNA Detection and Amplification Based on SPR Refractometric Scheme. The thiolated DNA probe immobilized sensor was mounted inside the SPR flow-cell for measurement. The surface was briefly flowed over with the HB buffer and the target miRNA-122 (in HB) was then injected into the chamber for hybridization with the surface immobilized DNA probes. To confirm the specificity of the D5H6 monoclonal antibody used in this study, the hybridization signal was amplified by the mab as shown in Figure S2a. The induced angular shifts for miRNA-122 binding and mab amplification were ∼0.1° and 0.2°, respectively. There were no significant angular changes in the control experiment using the nontarget miRNA-192 binding as shown in Figure S2b. Additionally, in situ kinetic measurements were carried out by monitoring the reflectivity at a fixed SPR incident angle (θ = 56°) as shown in Figure S3. The reflectivity change for mab enhanced target hybridization was ∼5-fold higher than that of the label-free binding. The nonspecific adsorption of the nontarget miRNA-192 and D5H6 mab was negligible, demonstrating the specific nature of the recognition of the antibody to the hybridized miRNA-122.

ultrasensitive detection of miRNA-23b using an imaging SPR instrument.11 Despite the exquisite sensitivity of this approach, in the fM range, its reliance on a surface initiated enzymatic amplification to introduce on the sensor surface polythymineAuNP conjugates limits its practical applicability. Conversely, streptavidin-coated AuNPs were successfully used as signal amplification tags for imaging SPR, allowing for the detection in erythrocyte lysate of a panel of miRNA associated with myelodysplastic syndrome in the pM sensitivity range and within 45 min.12 Toward increasing the intensity of the local perturbation of the evanescent field, graphene oxide-AuNP plasmonic hybrid molecular tags were designed and enabled detection of miRNA-141 in the low fM range.13 Despite their merits, these approaches based on AuNP amplification secondary tags are limited by either the necessity to prepare complex amplification tags or by the relative lack of sensitivity when using single AuNPs. The field enhancements associated with the binding of such secondary AuNP tags to the target miRNA sequences originates in the electromagnetic field coupling between propagating SPR and the localized surface plasmon of the AuNP as well as in the size/mass materials properties associated with AuNPs.14 With the aim of improving the signal enhancement associated with AuNP in SPR sensing schemes, we have demonstrated a novel approach based on surface plasmon resonance enhanced light scattering (SP-LS) and demonstrated its relevance for the detection of a proteinaceous biomarker, cardiac troponin I.15 We report here on the application of SPLS sensing for simple, rapid, and ultrasensitive detection of miRNAs. Using miRNA-122 as a model diagnostic biomarker, the absolute amount of attomoles could be detected in 50 μL of sample and within 30 min.

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EXPERIMENTAL SECTION

Materials. Chloroauric acid (HAuCl4·3H2O), trisodium citrate, hydroquinone, Tween 20, N-hydroxysuccinimide (NHS), N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), 6-mercapto-1-hexanol (MCH), diethylpyrocarbonate (DEPC), PerfectHyb hybridization buffer (HB), and phosphate buffer saline (PBS) were obtained from Sigma-Aldrich. The DNA and RNA sequences were synthesized by ThermoFisher as listed in Table 1. RNA-DNA

Table 1. Sequences of DNA and miRNA Used name

sequence (5′-3′)

Capture DNA probe miRNA-122 miRNA-192

HS-(CH2)6-CAA ACA CCA TTG TCA CAC TCC A UGG AGU GUG ACA AUG GUG UUU G CUG ACC UAU GAA UUG ACA GCC C

Figure 1. Schematic illustration of the SP-LS scheme for the detection of miRNA. 636

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Figure 2. Typical SPR angular spectra for refractometric SPR and SP-LS measurement for the (a) target miRNA-122 (100 nM) and (b) nontarget miRNA-192 (100 nM). Amplification of miRNA Hybridization in the SP-LS Scheme. Both refractometric and scattering schemes were monitored at the same time as illustrated in Figure 1. In the initial experiment, the sensor surface was flowed over with a 100 nM miRNA-122 solution for 15 min and then washed briefly with the buffer. The RNA-DNA hybridization signal was then amplified with the 55 nm AuNP@D5H6 mab for 10 min at different AuNP concentrations corresponding to OD of 2 and 10, as shown in Figure S4. Based on this initial study, the concentration of AuNP tags was fixed at OD = 10 for the subsequent studies. Simulations. In the simulation, three-dimensional Maxwell’s equations were solved using the finite element method (COMSOL Multiphysics). At the fixed incident light wavelength (λ0 = 632.8 nm), the dielectric function of gold was taken as 0.18 + 3.5i for the 48-nmthick gold film, and 0.574 + 2.89i for the 55-nm-diameter gold nanoparticles (AuNPs); the dielectric function of 2-nm-thick chromium was taken as 3.14 + 3.31i. The refractive indices for water, the prism LaSFN9, and the 10-nm-thick dielectric coating (i.e., mixture of antibody and DNA-RNA duplex) were 1.333, 1.845, and 1.458, respectively. A unit cell consisting of one nanoparticle sitting on multilayered SPR substrate was simulated by setting its lateral dimension as the pitch p, which defines the distance between nanoparticles. At the sides of the unit cell, Floquet periodic boundary condition was assumed in order to obtain the optical response of the whole array to a light source illuminating from an angle. At the top and bottom of the unit cell, we set a water perfectly matched-layer (PML) and a LaSFN9 PML to mimic the open boundaries, i.e., strongly absorb outgoing waves from the interior of a computational region without reflecting them back into the interior. An obliquely incident TM-polarized white light source (λ0 = 632.8 nm) was applied in the LaSFN9 domain. As the incident light wave strikes the array, its power will either be absorbed, reflected, or transmitted through the structure. The absorbed power was computed through the volume integration of the resistive heating in the gold nanoparticles, gold film, and chromium film. The reflected (or transmitted) power was calculated through the surface integration of the far-field power flow at the LaSFN9 (or water) side. The sum of calculated power of absorption, reflection, and transmission is checked against the incident power to ensure the accuracy of simulation. After solving the three-dimensional Maxwell’s equations, we could plot the percentage of power reflected back to the prism and compare it with the experimental measurement (results shown in Figure S6a). In addition, we could also estimate the percentage of power scattered from the AuNPs by taking the difference of the power transmission between the case of without AuNP and with AuNP and normalizing it to the area of the unit cell (results shown in Figure 4b).

probe immobilized on the sensor surface. Next, AuNPs conjugated with the D5H6 antibody (Covalab, France) that specifically recognizes and binds to the RNA*DNA hybrid duplexes are introduced to amplify the binding signal. RNA*DNA specific monoclonal antibodies (mabs) advantageously combine the selective recognition properties of antibodies and the high intrinsic selectivity of hybridization and have been successfully used in a number of biosensing approaches.17 For example, D5H6 mab binding to the RNADNA duplex provided significant signal amplification in refractometric SPR and yielded sensitivities for MiRNA-122 down to the low pM level.17 To confirm the affinity and specificity of the antibody used here to the hybridization of the miRNA-122 to the DNA probe, a refractometric SPR measurement was first conducted using miRNA-192 as a negative control (Supporting Information, Figure S2). A 5-fold signal enhancement was attained as a result of the specific binding of the antibody to the RNA*DNA duplex as compared to the label-free hybridization of miRNA-122 to it DNA probe (Figure S3). In order to achieve high sensitivity, the AuNP size used in SP-LS sensing is another important factor. According to the Rayleigh-Gans-Debye approximation, the dependence of the light scattering intensity, Is, is 4 orders of magnitude proportional to the particle diameter.18 Thus, 55 nm of AuNPs were selected as amplification tag to provide the desired high scattering intensity. Optimal biofunctionalization of the AuNP tags is also essential to ensure their efficient binding to the target.19,20 However, covalent attachment of monoclonal antibodies to AuNPs in this size range is not trivial. To build on our previous work, mixed PEG layers were used to achieve high functionalization without impacting on the colloidal stability of the AuNPs.16,21 Preparation of the antibody conjugated AuNPs was conducted ex situ as detailed in the Supporting Information (Figure S1). A high concentration (100 nM) of miRNA was first used to investigate the feasibility of the SP-LS assay for the amplification of miRNA (Figure S4). Following hybridization of miRNA-122 to the DNA probe, the D5H6 mab-AuNP conjugates were injected in the SPR flow cell at various optical densities (OD 2 and OD 10). It is worth noting that the miRNA hybridization step contributed 0.14% of reflectivity change; on the other hand, only negligible change in the scattered intensity occurred in response to the hybridization. Both the reflectivity and scattered intensity significantly increased in the presence of the D5H6-AuNP conjugates.

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RESULTS AND DISCUSSION The SP-LS principle for the detection of miRNAs is illustrated in Figure 1. The target miRNA binds to the DNA capture 637

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Figure 3. MiRNA-122 measurements and resulting calibration curves using 55 nm AuNP tags for (a and c) refractometric SPR and (b and d) SP-LS.

(Figure 3d). The LOD was determined as the intersection of the calibration curve with three times the standard deviation of the reflectivity/scattered intensity baseline noise (ΔR or ΔIs induced by the control miRNA-192).22,23 The calculated LODs for the refractometric and SP-LS schemes are 5 pM and 60 fM, respectively, allowing for the theoretical detection of miRNA122 at the absolute amounts of 175 attomoles (5 pM, 50 μL) and 2 attomoles (60 fM, 50 μL). To confirm the specificity of the method, these measurements were conducted for the target miR-122 in a high concentration of nontarget miRNA (1.0 nM of miR-192). A LOD of 300 fM was obtained, confirming the sensitivity and specificity of the assay. The sensitivity of SP-LS is therefore almost 2 orders of magnitude higher than that of the standard refractometric scheme which is in excellent agreement with our previous study with Troponin.15 The SPLS scheme shows better or comparable sensitivity for the detection of miRNA as compared with the previously reported SPR based methods listed in Table S1. SP-LS also provided 283-fold improvements in sensitivity as compared with previous studies for the detection of miRNA-122.17 However, it is important to note that these sensitivities, including the one obtained in this work, are obtained in ideal measurement conditions which do not take into account the biological noise in clinical samples associated with nonspecific adsorption of nontarget molecules. Finally, a simulation model (refer to Supporting Information for details) was developed using the finite element method to provide physical insight into the proposed SP-LS assay. The pitch parameter p (which denotes the interparticle distance of AuNPs) is used to correlate the surface coverage of the AuNP tags in the experimental measurements. As expected, the angular resonant angle increases with decreased pitches (i.e., higher surface density of AuNPs) (Figure S6). As shown in Figure 4a, it is revealed that the increased scattered intensity is attributed to the power scattered from AuNPs with the decrease of the pitch. The experimental observations shown in Figure 3b can be explained by simulated modeling confirming that the scattered intensity is boosted due to the large surface coverage of AuNPs resulting in higher concentrations of

The AuNP concentration equivalent to OD 10 provided rapid signal equilibrium and was therefore selected and used throughout this study. A synthetic miRNA-192 was used as a control to demonstrate the specificity of the proposed SP-LS miRNA assay. The miRNA-122 DNA capture probe was immobilized onto the SPR sensor, incubated with either the target or control miRNAs (100 nM) and the binding signal amplified using the D5H6-AuNP conjugates (OD = 10). As shown in Figure 2a, following the injection of the AuNP conjugates a small but detectable shift in the SPR resonant dip was observed for the miRNA-122 sample. A drastic boost in the scattered intensity was simultaneously detected in the SP-LS scheme. On the other hand, no detectable change was observed in the case of the miRNA-192 control for neither reflectivity nor scattering intensity. These initial results confirmed the specificity of D5H6-AuNP conjugates to the DNA-RNA hybrid duplexes. Next, the analytical performance of the SP-LS scheme was systematically investigated. To this end, in situ kinetic measurements were recorded for 10 min at the fixed angle of 56° with the concentrations of miRNA-122 ranging 0−100 nM and the recorded reflectivity measurements are shown in Figure 3a. The scattering spectra for the same experimental conditions are compiled in Figure 3b. The angular and scattering SPR spectra associated with the different concentrations of miRNA122 tested here are presented in Supporting Information (Figure S5). In the standard refractometric enhancement scheme, negligible reflectivity changes were detected for miRNA-122 concentration lower than 10 pM. A calibration curve for the reflectivity kinetic data was constructed (Figure 3c) and revealed that the signal is directly proportional to the concentration of the DNA-RNA duplexes. On the other hand, the scattering spectra for the SP-LS detection of miRNA-122 were normalized and compiled, indicating the feasibility of detecting subpicomolar concentrations. The processing methodology for the quantification of SP-LS data is detailed in the Supporting Information. The change of the scattered intensity (ΔIs) was plotted against the concentration of miRNA-122 to obtain the limit of detection 638

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CONCLUSION In conclusion, a simple, rapid, and sensitive signal amplification scheme based on SP-LS was demonstrated for the detection of miRNA. Using AuNPs conjugated with a mab with high affinity to RNA*DNA hybrid duplexes, SP-LS allowed the detection of miRNA-122 at subpicomolar concentrations within 30 min and a LOD of 60 fM was determined. The agreement between the experimental measurements and the simulation results confirms that SP-LS is a promising modality for the detection of diagnostic miRNA. Further studies are warranted to demonstrate the clinical utility of this novel approach. In addition, future efforts should be devoted to explore the effect of the sizes and shapes of the AuNP tag as well as to optimize the binding specificity of the AuNP tags in miRNA SP-LS assays performed in complex biological matrix such as serum toward further enhancing the sensitivity of this novel plasmonic sensing scheme.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00776. UV−vis, DLS, and SEM verification of as-synthesized AuNPs; SPR angular and kinetic spectra with regard to the detection of miRNA-122 and miRNA-192; angular/ scattered intensity spectra associated with the amplification using 55 nm AuNP@D5H6 mab in different target concentrations; simulation results; calibration curve of the detection of miRNA-122 diluted in miRNA-192 (1.0 nM) with 55 nm AuNP tags (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +61 8 83023689. Fax: + 61 8 8302 3683. ORCID

Benjamin Thierry: 0000-0002-6757-2842 Figure 4. (a) Simulated scattered power as a function of the incident angles for pitches p = 600, 800, and 1500 nm for 55 nm AuNPs. (b) Simulated scattered power for 36 and 55 nm AuNPs at p = 800 nm. (c) Simulated field enhancement distribution for 36 and 55 nm AuNPs at p = 800 nm. Inset: field enhancement distributions for 36 and 55 nm AuNPs at p = 800 nm.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project is supported by the National Health and Medical Research Council of Australia. M.P.M. also wants to thank University of South Australia for the grant of endeavour fellowship. This work was performed (in part) at the South Australian node of the Australian National Fabrication Facility under the National Collaborative Research Infrastructure Strategy to provide nano and microfabrication facilities for Australia’s researchers.

miRNA-122. Furthermore, the power scattered from two different AuNP sizes for p = 800 nm were also compared (Figure 4b). The power scattered from 55 nm of AuNPs is found to be 20-fold higher than that from 36 nm AuNPs, demonstrating the benefit of the employment of large particles for signal amplification tags in the SP-LS sensing scheme. In addition, the field enhancement distribution was also investigated with regard to the 36 and 55 nm AuNP tags. The correlation between the field enhancement and the scattering intensity induced by AuNPs has been discussed in our previous study.15 As shown in Figure 4c, 55 nm AuNPs induce more than 50% higher field enhancement in comparison to the 36 nm AuNPs at the interface of Au film/AuNPs, resulting in the significant improvement of scattering intensity observed for the 55 nm AuNPs.

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REFERENCES

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