Surface Plasmon Resonance Chip Surface Research Articles

Ultrasensitive Screening of Endocrine-Disrupting Chemicals Using a Surface Plasmon Resonance Biosensor with Polarization-Compensated Laser Heterodyne Feedback.

Developing an ultrasensitive and reliable device for continuous monitoring of various endocrine-disrupting chemicals (EDCs) is in high demand, yet it remains a significant challenge. Traditional label-free surface plasmon resonance (SPR) sensing relies on the interaction of the surface plasmon wave and the sensing liquid via intensity modulation, endowed with simple structure and easy-to-miniaturization, however suffering from inferior sensitivity and stability. Here, we propose a novel optical structure in which the frequency-shifted light of different polarization returned to the laser cavity to stimulate laser heterodyne feedback interferometry (LHFI), hence amplifying the reflectivity change caused by the refractive index (RI) variations on the gold-coated SPR chip surface, and the s-polarized light could be further used as a reference to compensate for the noise of the LHFI-amplified SPR system, resulting in nearly three orders of magnitude enhancement of the RI sensing resolution (5.9 × 10-8 RIU) compared to the original SPR system (2.0 × 10-5 RIU). To further boost intense signal enhancement, custom-designed gold nanorods (AuNRs), which were optimized by the finite-difference time-domain (FDTD) simulation, were used to generate localized surface plasmon resonance (LSPR). By exploiting the estrogen receptor as the recognition material, estrogenic active chemicals were detected with a 17β-estradiol/L detection limit of 0.004 ng, which is nearly 180-fold lower than that of the system without introducing AuNRs. The developed SPR biosensor is expected to be capable of screening various EDCs with universality by using several nuclear receptors, such as the androgen receptor and thyroid receptor, and will substantially accelerate the assessment of global EDCs.

An Ultrasensitive and Universal Surface Plasmonic Biosensor for Detection of Micropollutants in Aquatic Environments.

Simple yet ultrasensitive and accurate quantification of a variety of analytical targets by virtue of a universal sensing device holds promise to revolutionize environmental monitoring, medical diagnostics, and food safety. Here, we propose a novel optical surface plasmon resonance (SPR) system in which the frequency-shifted light of different polarizations returned the laser cavity to stimulate laser heterodyne feedback interferometry (LHFI), hence amplifying the reflectivity change caused by the refractive index (RI) variations on the gold-coated SPR chip surface. In addition, the s-polarized light was further used as the reference to compensate the noise of the LHFI-amplified SPR system, resulting in nearly 3 orders of magnitude enhancement of RI resolution (5.9 × 10-8 RIU) over the original SPR system (2.0 × 10-5 RIU). By exploiting nucleic acids, antibodies, and receptors as recognition materials, a variety of micropollutants were detected with ultralow detection limits, ranging from a toxic metal ion (Hg2+, 70 ng/L) to a group of commonly occurring biotoxin (microcystins, 3.9 ng microcystin-LR/L) and a class of environmental endocrine disruptors (estrogens, 0.7 ng 17β-estradiol/L). This sensing platform exhibits several distinct characteristics, including dual improvement of sensitivity and stability and common-path optical construction without needing optical alignment, demonstrating a promising avenue toward environmental monitoring.

Molecular imprinted nanoparticle assisted surface plasmon resonance biosensors for detection of thrombin

In this study, we designed a surface plasmon resonance (SPR) biosensors based on molecular imprinting to detect low amounts of thrombin directly in a short time, both in thrombin aqueous solutions and in patient serum samples. For this purpose, the thrombin imprinted and non-imprinted SPR biosensors were prepared by integrating the synthesized thrombin imprinted and non-imprinted nanoparticles on the allyl mercaptan modified gold SPR chip surface. The kinetic studies were performed with a thrombin concentration range from 0.1 to 400 pM with a detection limit of approximately 0.017 pM in thrombin aqueous solution. Morever, the limit of detection was found to be 0.033 pM in patient serum samples. When the selectivity of the thrombin imprinted SPR biosensors was compared with the competing molecules as bovine serum albumin and lysozyme, it was showed that the thrombin was 4.58 times and 3.99 times more selective than the bovine serum albumin and lysozyme molecules, respectively. The shelf life and reusability of the designed SPR biosensor showed that there was a 12.93% decrease in the performance according to the kinetic analyzes that performed after 8 months. In addition, thrombin detection from the patient serum samples was performed using both SPR biosensor and the enzyme-linked immunosorbent assay methods, and were calculated recoveries.

A surface plasmon resonance database for better experimental planning and execution

Surface plasmon resonance (SPR) is a label-free biophysical technique that is widely used to investigate biomolecular interactions and a very powerful tool useful in different stages of small molecule drug development and antibody characterization. Direct molecular binding on the SPR chip surface is recorded as a response in real time. Several experimental variables, including buffer composition, type of sensor chip, coupling chemistry of molecules on the sensor surface, and surface regeneration conditions, contribute to quality and accuracy of the SPR data.

Interfacing Live Cells with Surfaces: A Concurrent Control Technique for Quantifying Surface Ligand Activity.

Surface ligand activity is a key design parameter for successfully interfacing surfaces with cells─whether in the context of in vitro investigations for understanding cellular signaling pathways or more applied applications in drug delivery and medical implants. Unlike other crucial surface parameters, such as stiffness and roughness, surface ligand activity is typically based on a set of assumptions rather than directly measured, giving rise to interpretations of cell adhesion that can vary with the assumptions made. To fill this void, we have developed a concurrent control technique for directly characterizing in vitro ligand surface activity. Pairs of gold-coated glass chips were biofunctionalized with RGD ligand in a parallel workflow: one chip for in vitro applications and the other for surface plasmon resonance (SPR)-based RGD activity characterization. Recombinant αVβ3 integrins were injected over the SPR chip surface as mimics of the cellular-membrane-bound receptors and the resulting binding kinetics parameterized to quantify surface ligand activity. These activity measurements were correlated with cell morphological features, measured by interfacing MDA-MB-231 cells with the in vitro chip surfaces on the live cell microscope. We demonstrate how the interpretation of a cell phenotype based on direct activity measurements can vary markedly from interpretations based on assumed activity. The SPR concurrent control approach has multiple advantages due to the fact that SPR is a standardized technique and has the sensitivity to measure ligand activity across the most relevant range of extracellular surface densities, while the in vitro chip design can be used with all commonly used light microscopy modalities (e.g., phase contrast, DIC, and fluorescence) so that a wide range of phenotypic and molecular markers can be correlated to the ligand surface activity.

Real‐Time Detection of Fibrinogen via Imprinted Recognition Sites

AbstractIn this study, the fibrinogen imprinted surface plasmon resonance (SPR) biosensor was developed for the monitoring of fibrinogen levels in patient serum samples using a microcontact imprinting method. The fibrinogen imprinted and non‐imprinted polymeric films on the SPR chip surface were prepared by the microcontact imprinting approach and characterized by ellipsometer, contact angle measurements, and Fourier transform infrared spectroscopy. The limit of detection and quantification values of the fibrinogen imprinted SPR biosensors in the linear range of 0.05–9.0 mg/mL were 0.00066 and 0.00219 mg/mL, respectively. In all kinetic analyzes, the response time was 11 min for equilibration, adsorption, and desorption cycles. The selectivity studies of SPR biosensors were performed in the presence of fibrinogen, myoglobin, hemoglobin, and immunoglobulin G in a competitive manner. For the validation studies of the fibrinogen imprinted SPR biosensor, the fibrinogen level of diluted patient serum samples was determined by enzyme‐linked immunosorbent assay (ELISA). The experimental results show that the serum samples of patients in high and low‐risk groups were identified with the recovery of about % 97–99 according to both SPR biosensor and ELISA analysis results.

Preparation of surface plasmon resonance-based nanosensor for curcumin detection.

In this study, the curcumin imprinted and the non-imprinted poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-tryptophan) (poly(HEMA-MATrp)) nanoparticle based surface plasmon resonance (SPR) nanosensors were prepared for the detection of curcumin and characterized by zeta-size analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. After, the curcumin imprinted and the non-imprinted nanoparticles are attached on the surface of SPR chips. The curcumin imprinted and the non-imprinted SPR nanosensors are characterized by using atomic force microscope, ellipsometer, and contact angle measurements. Kinetic studies were carried out with curcumin aqueous solution at a concentration range of 0.01-150 mg/L using the curcumin imprinted and the non-imprinted SPR nanosensors. In all kinetic analysis, the response time is 14 min for equilibration, adsorption, and desorption cycles. The limit of detection and limit of quantification for the curcumin imprinted SPR nanosensors was 0.0012 mg/L and 0.0040 mg/L, respectively. The validity of the curcumin imprinted SPR nanosensors in real samples was carried out using liquid chromatography-tandem mass spectrometry (LC-MS).

Design and preparation of imprinted surface plasmon resonance (SPR) nanosensor for detection of Zn(II) ions

A novel Zn(II) ions imprinted poly (2-hydroxyethyl Methacrylate-N-methacryloyl-(L)-histidine methyl ester) poly(HEMAH) surface plasmon resonance (SPR) nanosensor were designed for detection of Zn(II) ions in aqueous solution and artificial plasma providing a low cost, rapid and reliable results compared to other techniques such as atomic absorption spectroscopy, inductively coupled plasma-mass spectrometer, X-ray fluorescence with synchrotron radiation. Zn(II) ions imprinted nanofilm on the SPR chip surface was synthesized by bulk polymerization. Characterization of Zn(II) ions imprinted nanosensor was performed by contact angle measurement, atomic force microscopy (AFM), ellipsometry and Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR). Designed nanosensor was applied for selective detection of Zn(II) ions in aqueous solution within the range of 0.5–1.0 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as 0.19 and 0.64 ng/mL, respectively. Association kinetics analysis, Scatchard, Langmuir, Freundlich, Langmuir–Freundlich, Tempkin and Dubinin-Radushkevich isotherms were analyzed to the experimental data in order to identify the adsorption behavior. The selectivity of the SPR nanosensor was examined by using competitive metal ions such as Cd(II), Cu(II), Pb(II), and Fe(II). To evaluate the imprinting effect of Zn(II) ions imprinted (MIP) and non-imprinted (NIP) nanosensor was also prepared as the control. Repeatability of the response signal was tested by four times adsorption–desorption–regeneration cycle.

Direct quantification of cancerous exosomes via surface plasmon resonance with dual gold nanoparticle-assisted signal amplification

Sensitive detection of cancerous exosomes is critical to early diseases diagnosis and prognosis. Herein, a sensitive aptasensor was demonstrated for exosomes detection by surface plasmon resonance (SPR) with dual gold nanoparticle (AuNP)-assisted signal amplification. Dual nanoparticle amplification was achieved by controlled hybridization attachment of AuNPs resulting from electronic coupling between the Au film and AuNPs, as well as coupling effects in plasmonic nanostructures. By blocking the Au film surface with 11-Mercapto-1 -undecanol (MCU), nonspecific adsorption of AuNPs onto the SPR chip surface was suppressed and regeneration of the SPR sensor was realized. This method was highly sensitive and we have achieved the limit of detection (LOD) down to 5 × 103 exosomes/mL, which showed a 104-fold improvement in LOD compared to commercial ELISA. Moreover, the SPR sensor had the capability to differentiate the exosomes secreted by MCF-7 breast cancer cells and MCF-10A normal breast cells. Furthermore, the SPR sensor could effectively detect the exosomes in 30% fetal bovine serum. The work provides a sensitive and efficient quantification approach to detect cancerous exosomes and offers an avenue toward future diagnosis and comprehensive studies of exosomes.

Molecularly imprinted based surface plasmon resonance nanosensors for microalbumin detection

Human serum albumin (HSA) is a major blood plasma protein also found in urine where its existence may be a marker of some types of liver or kidney dysfunction. Herein, we fabricated a novel surface plasmon resonance (SPR) nanosensor for selective, sensitive, and label-free microalbumin detection both in aqueous and urine sample solutions. First, HSA-imprinted nanoparticles were synthesized, which consist of ethylene glycol dimethacrylate and N-methacryloyl-L-leucine methyl ester as a cross-linker and functional monomer. The nanoparticles were characterized by zeta-size and scanning electron microscope analyses and were dropped onto the SPR chip surface to make HSA sensitive nanosensor. Characterization studies of HSA-imprinted SPR chip were carried out by atomic force microscopy, Fourier-transform infrared spectroscopy, contact angle, and ellipsometer. The limit of detection and limit of quantification values of HSA-imprinted SPR nanosensor were calculated as 0.7 pM and 1.9 pM for the concentration range of 0.15–500 nM. Selectivity studies of HSA-imprinted SPR nanosensor were achieved with hemoglobin and transferrin proteins which were chosen as competitor molecules. HSA-imprinted SPR nanosensor was displayed highly selective and sensitive to HSA.

Improving nanoparticle-enhanced surface plasmon resonance detection of small molecules by reducing steric hindrance via molecular linkers

Ultrasensitive Surface Plasmon Resonance (SPR) detection of small molecules can be achieved using nanoparticles to both enhance the SPR signals and pre-concentrate low levels of target analytes in the sample. However, the short effective penetration depth of the SPR evanescent field, and steric hindrance of binding when immobilizing small molecules on surfaces, limits the applicability of using relatively large nanoparticles (≥100 nm) for SPR detection. To overcome the issues of steric hindrance, this paper investigates the role of the molecular linkers to tether both the antibodies to the magnetic nanoparticles, and to bind the small molecules to the surface of the SPR chips. By extending the distance of the small molecule (progesterone) away from the SPR chip surface and improving the antibody orientation on the large magnetic nanoparticles, a sensitive SPR detection for progesterone was achieved in buffer (0.013 ng mL−1). The results of the SPR assay for progesterone in milk were in good correlation with ELISA results, and could be used to verify the onset of estrus in cows.

Detection of ciprofloxacin through surface plasmon resonance nanosensor with specific recognition sites

The novel nanosensor based on Surface Plasmon Resonance (SPR) was developed for sensitive and selective detection of ciprofloxacin via molecularly imprinted nanoparticles (MIP/NPs). NPs were synthesized through miniemulsion polymerization technique with methacrylic acid as a functional monomer. FTIR, SEM, zetasizer and contact angle measurements were used for the characterization of MIP/NPs. After modification the SPR chip surface, the nanosensor was used for detection of ciprofloxacin in aqueous solution. According to selected concentration range, the correlation coefficient and limit of detections were obtained as 0.993 (R2) and 3.21 and 7.1 ppb in ultrapure water and SWW, respectively. Association kinetic analysis, Scatchard, Freundlich, Langmuir and Freundlich-Langmuir isotherms were also performed on the data to investigate adsorption behaviour of ciprofloxacin on the surface of nanosensor. Tetracycline and enrofloxacin were used as competitor agents to examine the selectivity of nanosensor. Performance of the SPR nanosensor was also investigated by using synthetic wastewater (SWW) for detection of CPX. The reusability of nanosensor was investigated and good repeatability was obtained with 5.81% RSD. As a result, selective, simple and low-cost method to detect ciprofloxacin in aqueous solution was developed by combining MIP/NPs and SPR.

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody

BackgroundG-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens. MethodsSPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures. ResultsThe use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability. ConclusionsOptimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells. General significanceThe heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface.

Graphene oxide linking layers for highly sensitive SPR biosensing of small molecules

The development of graphene oxide (GO) interfaces can significantly improve the performance of modern surface plasmon resonance (SPR) biosensors. GO linking layers have high binding capacity for a wide range of chemical and biological ligands owing to large surface area of GO and its unique biochemical properties. Here, we compared binding capacities of GO linking layers with different thicknesses and widely-used hydrogel linking layers based on carboxymethylated dextran (CMD). SPR measurements corresponding to the activation of carboxyl groups on the surface of SPR chips showed that the binding capacity of a 50-nm-thick GO linking layer is 30-times higher than for 150-nm-thick CMD layer, which results in enhanced biosensing sensitivity of GO chips and can lead to novel applications for SPR biosensing.

Site-Specific Immobilization of the Peptidoglycan Synthase PBP1B on a Surface Plasmon Resonance Chip Surface.

Surface plasmon resonance (SPR) is one of the most powerful label‐free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin‐binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin‐coated chip surface (a β‐lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate‐bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N‐terminal transmembrane helix, and hence immobilization at the C‐terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper‐free click chemistry at an azide incorporated in the N terminus. In a proof‐of‐principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well‐characterized interaction of PBP1B with LpoB. The site‐specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology‐dependent interactions of any (membrane) protein.

A Portable Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella typhimurium

Here, the rapid detection of Salmonella typhimurium by a portable surface plasmon resonance (SPR) biosensor in which the beam from a diode laser is modulated by a rotating mirror is reported. Using this system, immunoassay based on lipopolysaccharides (LPS)-specific monoclonal anti-Salmonella antibody was performed. For the purpose of orientation-controlled immobilization of antibodies on the SPR chip surface, the cysteine-mediated immobilization method, which is based on interaction between a gold surface and a thiol group (-SH) of cysteine, was adopted. As a result, using the portable SPR-based immunoassay, we detected S. typhimurium in the range from 10^7 CFU/mL to 10^9 CFU/mL within 1 hour. The results indicate that the portable SPR system could be potentially applied for general laboratory detection as well as on-site monitoring of foodborne, clinical, and environmental agents of interest.

Sensitive detection of aflatoxin B1 molecules on gold SPR chip surface using functionalized gold nanoparticles

Due to the warm and favourably humid climate of Southern Hungary, the maize is one of the most important crops. The protection against crop damage caused by fusarium and Aspergillus species is essential. Detection of aflatoxin B1 (AFB1) molecules in cereal crops by selective sensors is important, while they can cause serious diseases in humans and animals if they enter the food chain. Our main objective was to develop selective AFB1 sensor with increased sensitivity applying βCD-functionalized gold nanoparticles (AuβCD NPs) in surface plasmon resonance (SPR) measuring apparatus. The nanoparticles ca. 10 nm in diameter were prepared in the presence of thiol-modified cyclodextrin. The adsorption isotherms of AFB1 on bare, thiol-modified cyclodextrin and AuβCD NPs covered Au film surface were calculated using SPR platform. The AFB1 concentration can be quantitatively determined in the 0.001–23.68 ng/mL range. The AuβCD NPs were found to be highly sensitive and exhibited a remarkably low limit of detection (LOD; 1 pg/mL) without using other analytical reagents.

Fatty acid binding to CD36 affects oxLDL uptake

We investigated the hypothesis that interactions of unesterified fatty acid (FA) with the plasma membrane protein CD36 affect uptake of oxLDL. Because the binding of natural FA to CD36 have not been systematically studied, we first characterized FA binding to CD36 by the biophysical method surface plasmon resonance (SPR). After covalently linking the extracellular domain of CD36 to an SPR chip surface, aqueous FA (solubilized in cyclodextrin) was pulsed across the surface, generating association and dissociation binding curves. We studied structurally different long chain FA: a saturated (palmitic acid); unsaturated (oleic acid); trans‐oleic acid; (elaidic); a polyunsaturated [docosahexaenoic acid (DHA)], and an oxidized FA [9S‐hydroxy‐10E,12Z‐octadecadienoic acid (HODE)]. With the exception of HODE, SPR detected FA binding to CD36, and showed that multiple sites are present with rapid association and dissociation kinetics that were similar to HSA. To study the role in oxLDL uptake of these FA, we used a fluorescent oxLDL (Dii‐oxLDL) live‐cell assay with confocal microscopy imaging. CD36‐mediated uptake in serum‐free media was very low but greatly increased with serum. Addition of FA in serum‐free media increased oxLDL binding and uptake to levels found with serum, and affected CD36 plasma membrane (PM) distribution. Binding/uptake of oxLDL exhibited dose dependency of added FA, with the exception of DHA, which binds avidly to CD36 but did not activate uptake. HODE, which binds poorly to CD36, did not affect oxLDL uptake. The high affinity FA binding to CD36 has important implications that binding at some or all sites could affect its conformation, binding of other ligands, and functional properties.

Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments

The interaction between kynurenic acid (KYNA) and two peptide fragments (ca. 30 residues) of Human Glutamate Receptor 201–300 (GluR1) using surface plasmon resonance (SPR) spectroscopy was investigated. Because of the medical interest in the neuroscience, GluR1 is one of the important subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). AMPARs are ionotoropic glutamate receptors, which are mediating fast synaptic transmission and are crucial for plasticity in the brain. On the other hand, KYNA has been suggested to have neuroprotective activity and it has been considered for apply in therapy in certain neurobiological disorders. In this article the adsorption of the GluR1201–230 and GluR1231–259 peptides were studied on gold biosensor chip. The peptides were chemically bonded onto the gold surface via thiol group of l-cysteine resulted in the formation of peptide monolayer on the SPR chip surface. Because the GluR1231–259 peptide does not contain l-cysteine the Val256 was replaced by Cys256. The cross sectional area and the surface orientation of the studied peptides were determined by SPR and theoretical calculations (LOMETS) as well. The binding capability of KYNA on the peptide monolayer was studied in the concentration range of 0.1–5.0mM using 150mM NaCl ionic strength at pH 7.4 (±0.02) in phosphate buffer solutions. In order to determine the binding enthalpy the experiments were carried out between +10°C and +40°C. The heat of adsorption was calculated by using adsorption isotherms at different surface loading of KYNA on the SPR chip.

Dual nanoparticle amplified surface plasmon resonance detection of thrombin at subattomolar concentrations.

A novel dual nanoparticle amplification approach is introduced for the enhanced surface plasmon resonance (SPR) detection of a target protein at subattomolar concentrations. Thrombin was used as a model target protein as part of a sandwich assay involving an antithrombin (anti-Th) modified SPR chip surface and a thrombin specific DNA aptamer (Th-aptamer) whose sequence also includes a polyadenine (A30) tail. Dual nanoparticle (NP) enhancement was achieved with the controlled hybridization adsorption of first polythymine-NP conjugates (T20-NPs) followed by polyadenine-NPs (A30-NPs). Two different nanoparticle shapes (nanorod and quasi-spherical) were explored resulting in four different NP pair combinations being directly compared. It was found that both the order and NP shape were important in optimizing the assay performance. The use of real-time SPR measurements to detect target concentrations as low as 0.1 aM is a 10-fold improvement compared to single NP-enhanced SPR detection methods.