Nanoparticle Enhancement Cascade for Sensitive Multiplex Measurements of Biomarkers in Complex Fluids with Surface Plasmon Resonance Imaging

Abstract

There is a large unmet need for reliable biomarker measurement systems for clinical application. Such systems should meet challenging requirements for large scale use, including a large dynamic detection range, multiplexing capacity, and both high specificity and sensitivity. More importantly, these requirements need to apply to complex biological samples, which require extensive quality control. In this paper, we present the development of an enhancement detection cascade for surface plasmon resonance imaging (SPRi). The cascade applies an antibody sandwich assay, followed by neutravidin and a gold nanoparticle enhancement for quantitative biomarker measurements in small volumes of complex fluids. We present a feasibility study both in simple buffers and in spiked equine synovial fluid with four cytokines, IL-1β, IL-6, IFN-γ, and TNF-α. Our enhancement cascade leads to an antibody dependent improvement in sensitivity up to 40 000 times, resulting in a limit of detection as low as 50 fg/mL and a dynamic detection range of more than 7 logs. Additionally, measurements at these low concentrations are highly reliable with intra- and interassay CVs between 2% and 20%. We subsequently showed this assay is suitable for multiplex measurements with good specificity and limited cross-reactivity. Moreover, we demonstrated robust detection of IL-6 and IL-1β in spiked undiluted equine synovial fluid with small variation compared to buffer controls. In addition, the availability of real time measurements provides extensive quality control opportunities, essential for clinical applications. Therefore, we consider this method is suitable for broad application in SPRi for multiplex biomarker detection in both research and clinical settings.

Low-Fouling Substrates for Plasmonic Sensing of Circulating Biomarkers in Biological Fluids

“…In this line, spiked equine synovial fluid was used to detect cytokines by means of an SPRi immunoassay [26]. The assay consisted of an antibody sandwich cascade with subsequent addition of neutravidin and biotinylated gold nanoparticles as signal amplification.…”

“…Therefore, current plasmonics applications are taking advantage of the functionalization of nanomaterials to improve the detection of a broad range of biomarkers, avoiding time-consuming multistep procedures and highly demanding biofluid requirements [18]. Particularly, novel plasmonic approaches based on the chemical activation of different types of nanoparticles have been applied to the determination of a broad range of biomarkers from circulating tumor-associated nucleic acids to living cells, proteins or exosomes [7,[26][27][28]. In the same vein, the development of nanostructures also relies on the fabrication of plasmonic surfaces based on optical antenna structures such as arrays of nanoholes, nanoslits, nanostars or nanodiscs [29][30][31].…”

Digital Duplex Homogeneous Immunoassay by Counting Immunocomplex Labeled with Quantum Dots

“…Typical examples are various array-based immunoassays, such as electrode arrays, 18 nanowire arrays, 22 microfluidic channel arrays, 23,24 nanopore arrays, 25 and dot arrays of surface-enhanced Raman scattering (SERS) substrates. 26 Another was a destined tag-labeled antibody for a specific immunocomplex. 21,27−32 Nanoplasmonic particles, 27 SERS tags, 28,29 electrochemical tags, 30 QDs, 31 organic fluorescent dyes, 30 and magnetic beads 32 were exploited for these assays.…”

“…To recognize immunocomplexes in heterogeneous multiplex immunoassays, researchers usually use two strategies. One is the spatial position, where the signal is detected. − ,− Specific domains of the sensor are decorated with destined antibodies, and thus, immunocomplexes can be easily identified by the signal location. Typical examples are various array-based immunoassays, such as electrode arrays, nanowire arrays, microfluidic channel arrays, , nanopore arrays, and dot arrays of surface-enhanced Raman scattering (SERS) substrates .…”

Advances in nanoplasmonic biosensors for clinical applications

“…Therefore, the rapid and real-time detection conferred by plasmonic biosensors has triggered the number of troponin applications in recent years. Most of the strategies rely on immunoassays using either SPR or LSPR biosensor platforms.SPR detection methods commonly involve gold nanoparticles secondary signal enhancement.Pawula et al describe Troponin T(tnT) detection in serum samples by using a SPR biosensor 105.…”