ACS Nano. 2015 April; 9(4): 4173-4181
Multiplex Serum Cytokine Immunoassay Using Nanoplasmonic Biosensor Microarrays
Chen P, Chung MT, McHugh W, Nidetz R, Li Y, Fu J, Cornell TT, Shanley TP, Kurabayashi K.
Department of Mechanical Engineering, University of Michigan, Ann, Arbor, 48109, USA
Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, 48109, USA
Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making (e.g. critically-ill patients) impractical. Without such assays, immune monitoring is even virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders as access to their blood in large quantities is prohibited. Localized surface plasmon resonance (LSPR)-based microfluidic optical biosensing is a promising approach to fill this technical gap as it could potentially permit real-time refractometric detection of biomolecular binding on a metallic nanoparticle surface and sensor miniaturization, both leading to rapid and sample-sparing analyte analysis. Despite this promise, practical implementation of such a microfluidic assay for cytokine biomarker detection in serum samples has not been established primarily due to the limited sensitivity of LSPR biosensing. Here, we developed a high-throughput, label-free, multi-arrayed LSPR optical biosensor device with 480 nanoplasmonic sensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokines in a complex serum matrix on a single device chip while overcoming technical limitations. The device was fabricated using easy-to-implement, one-step microfluidic patterning and antibody conjugation of gold nanorods (AuNRs). Scanning the scattering light intensity across the microarrays of AuNR ensembles with dark-field imaging optics, our LSPR biosensing technique allowed for high-sensitivity quantitative cytokine measurements at concentrations down to 5 – 20 pg/mL from a 1 mL serum sample. Using the nanoplasmonic biosensor microarray device, we demonstrated the ability to monitor the inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking the time-course variations of their serum cytokines. The whole parallel on-chip assays, which involved the loading, incubation, and washing of samples and reagents, and ten-fold replicated multi-analyte detection for each sample using the entire biosensor arrays, were completed within 40 minutes.
Over the past years, researchers have perceived nanoplasmonics-based biomolecular binding assay enabled by localized surface plasmon resonance (LSPR) as a label-free optical biosensing technique emerging with recent advances in nanotechnology. Conventional methods for multiplex disease biomarker detection in clinical diagnosis are based on sandwich immunoassays. Multiplex immunoassay using our nanoplasmonic biosensor microarray holds promise to eliminate shortcomings of the conventional methods, such as cross-reaction between multiple secondary antibodies, a limited number of labeling agents, time-consuming labeling processes, and an onset of undesirable photochemical reaction or photobleaching upon excitation, while permitting sensor miniaturization and integration. However, implementation of LSPR biosensing for protein biomarker detection in complex clinical samples has been prohibited primarily by a lack of balance between assay sensitivity and speed. We have successfully overcome these challenges by synthesizing stripe-shaped assemblies of bioconjugated gold nanorod particles using microfluidic flow patterning, preforming massively parallel on-chip binding assay with a biosensor-integrated optofulidic platform, and implementing high-throughput LSPR imaging for AuNR microarrays.
Figure 1. a) Schematic of the LSPR nanoplasmonic microarray chip. b) Dark-field and scanning electron microscopy (SEM) images of AuNR biosensors fabricated and functionalized using a one-step microfluidic patterning technique assisted by electrostatic attractive interactions within microfluidic channels. c) The principle of the LSPR microarray imaging method.
Figure 1a shows our LSPR microarray biochip consisting of eight parallel microfluidic channels. These channels run orthogonal to six meandering stripes of antibody-functionalized AuNR ensembles with ten turns on a glass substrate. Specific antibodies were conjugated to the patterned AuNR microarrays using thiolated crosslinker and EDC/NHC chemistry. Each microfluidic channel holds 250 nL in volume and has inlet and outlet ports for reagent loading and washing. The chip design gives rise to an array of 480 stripe-shaped LSPR biosensing spots (25 m x 200 m) on the entire chip.
A scanning electron microscopy (SEM) image in Figure 1b validates that the LSPR sensing spots were coated with a relatively disordered monolayer of AuNR particles at a surface number density of ~ 1 particles per 2.56 mm2, which corresponded to an average particle-to-particle distance greater than 200 nm. The dispersed distribution of AuNRs effectively eliminates electromagnetic (EM) couplings between neighboring nanoparticles in the LSPR biosensing ensembles. As a result, the multi-arrayed LSPR sensor performance was solely determined by the superimposition of the optical characteristics of individual nanoparticles while uninfluenced by the nanoparticle arrangement. It follows that the ensemble of ~2,000 plasmonically uncoupled AuNRs on each sensor spot could yield a scattering spectrum with a distinct resonance without particle-to-particle EM interferences, which led to the high sensitivity of our device.
We employed dark-field imaging that scans the scattering light intensity across the LSPR biosensing spots for signal detection as illustrated in Figure 1c. Analyte molecules were introduced to antibody-functionalized AuNR LSPR biosensors. Binding of the analyte molecules to the receptors induced a redshift and scattering intensity change for each AuNR biosensor. This intensity change across the ensemble of AuNR biosensors was collectively imaged via the characteristic optical band (grey area) using electron multiplying charge coupled device (EMCCD)-coupled dark-field microscopy.
Our study successfully achieved multiplexed quantification of six cytokines (IL-2, IL-4, IL-6, IL-10, INF-a, and INF-g) in human blood serum as shown in Figure 2a with a limit of detection of ~10 pg/mL and a sample volume as small as 1 mL simultaneously using the 480 on-chip gold nanorod biosensing spots. Figure 2b shows our label-free biosensor’s ability to monitor the analyte binding kinetics in real-time, which allowed us to ensure the assay reached the equilibrium within 15 min. As a result, the total assay time became as short as 30 -40 min. Furthermore, our study validated our multiplex immunoassay by obtaining an excellent correlation with ELISA-based results.
Figure 2. a) Calibration curves of TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10 obtained from the LSPR microarray intensity mapping. The dashed lines represent the sigmoidal curves fitted to data points. b) Real-time AuNR microarray signals during multiplexed cytokine immunoassay, involving sample pre-loading, sample incubation, signal equilibration, and sample washing. c) Five-day cytokine concentration variations measured by the LSPR microarray assay for serum samples extracted from four post-cardiopulmonary bypass surgery pediatric patients.
Repair of congenital heart defects, the most common birth defect in the United States, necessitates open-heart surgery using cardiopulmonary bypass (CPB) to supplant heart-lung function during surgery. Blood contact with the artificial surfaces of the CPB circuit is known to elicit a substantial inflammatory response in the immediate post-operative period that is normally restored to pre-operative levels within 48 hours after surgery. We collected serum samples prior to surgery (Pre) and on post-operative days one (D1), two (D2), three (D3), and four (D4) from two neonates undergoing congenital heart surgery with CPB and used our LSPR microarray immunoassay to quantify circulating serum cytokine levels in these samples as shown in Figure 2c. We observed increased levels of both IL-6 and IL-10 on D1 following CPB in both patients although they were expressed at different levels. Post-operative heightening was also observed for IL-2, IL-4, TNF-a, and INF-g in these patients on D1 or D2. The elevated cytokine expression was followed by a return to pre-operative levels of all the measured cytokines on D3 and D4. We also observed a very similar pattern to those previously reported where elevations in patient’s serum cytokine levels most commonly return to pre-surgical levels within 48 hours of surgery. Such information has proven valuable since it is known that very high and/or prolonged expression of both pro-inflammatory (e.g. IL-6) and anti-inflammatory (e.g. IL-10) cytokines are associated with the acute immune dysfunction following cardiopulmonary bypass and can predict worse outcomes. This pattern of expression is thought to result from the host’s misregulated compensatory effort to counter an initial pro-inflammatory response that occurs perioperatively as a result of cardiopulmonary bypass. Most importantly, the LSPR microarray assay has been shown to be able to detect variable degrees of responses in the four subjects after CPB.
Impact of the study:
The demonstrated label-free multiplexed immunoassay using our nanoplasmonic biosensing platform can serve as a powerful tool for the detection of a wide spectrum of biomarkers relevant to human disease screening. The platform can also extend into the basic science arena providing small-volume, rapid detection of proteins from cell media or animal serum. The assay process is repeatable and easy to implement, which can readily be replicated by other research groups. To our best knowledge, this study is the first to demonstrate practical implementation of LSPR-based nanobiosensing for a clinically relevant setting. We believe that our method will open the door for personalized immunomodulatory therapy of systemic inflammatory diseases, enabled by rapid cytokine-based immune status monitoring (1). More recently, our platform has also been used for dynamic in-situ monitoring of the cytokine secretion function of T cells under drug exposure (2). The data obtained from the multi-cytokine secretion profiles may provide a comprehensive picture of the time-varying cellular functional state during pharmacologic regulation of the immune cells.
- Chen P, Huang NT, Chung MT, Cornell TT, Kurabayashi K, 2015 Label-free cytokine micro-and nano-biosensing towards personalized medicine of systemic inflammatory disorders. Advanced Drug Delivery Reviews 95: 90-103
- Oh BR, Chen P, Nidetz R, McHugh W, Fu J, Shanley TP, Cornell TT, Kurabayashi K, 2016 Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure. ACS Sensors DOI: 10.1021/acssensors.6b00240
Acknowledgements: This work was supported by the National Institute of Health (grant no.R01 HL119542-01A1), National Science Foundation (grant no. CBET 1263889), and the National Center for Advancing Translational Sciences he Michigan Institute for Clinical & Health Research (grant no. 2UL1TR000433).
Katsuo Kurabayashi, Ph.D.
Professor and Associate Chair
Department of Mechanical Engineering
2380B G.G. Brown Lab
University of Michigan
Ann Arbor, MI 48109-2125
Pengyu Chen, Ph.D.
Department of Materials Engineering
Auburn, AL 36849