Single Nanoparticle Detection for Multiplexed Protein Diagnostics with Attomolar Sensitivity in Serum and Unprocessed Whole Blood

Abstract

Although biomarkers exist for a range of disease diagnostics, a single low-cost platform exhibiting the required sensitivity, a large dynamic-range and multiplexing capability, and zero sample preparation remains in high demand for a variety of clinical applications. The Interferometric Reflectance Imaging Sensor (IRIS) was utilized to digitally detect and size single gold nanoparticles to identify protein biomarkers in unprocessed serum and blood samples. IRIS is a simple, inexpensive, multiplexed, high-throughput, and label-free optical biosensor that was originally used to quantify biomass captured on a surface with moderate sensitivity. Here we demonstrate detection of β-lactoglobulin, a cow’s milk whey protein spiked in serum (>10 orders of magnitude) and whole blood (>5 orders of magnitude), at attomolar sensitivity. The clinical utility of IRIS was demonstrated by detecting allergen-specific IgE from microliters of characterized human serum and unprocessed whole blood samples by using secondary antibodies against human IgE labeled with 40 nm gold nanoparticles. To the best of our knowledge, this level of sensitivity over a large dynamic range has not been previously demonstrated. IRIS offers four main advantages compared to existing technologies: it (i) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, (ii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, (iii) detects protein targets with conjugated very small nanoparticle tags (~40 nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and (iv) utilizes components that make the instrument inexpensive, robust, and portable. These features make IRIS an ideal candidate for clinical and diagnostic applications.

Digital Detection of Nanoparticles: Viral Diagnostics and Multiplexed Protein and Nucleic Acid Assays

“…Direct detection of protein biomarkers with attomolar sensitivity without the need of sample preparation can allow performing diagnostic tests at the point of care. We have demonstrated quantitation of allergen-specific IgE from unprocessed finger prick volume of human blood [4]. We have also conducted proof of principle experiment showing that SP-IRIS can screen for nucleic acid based biomarkers.…”

“…We have demonstrated the simultaneous detection of multiple viruses in serum or whole blood as well as in samples contaminated with high levels of bacteria [3]. Single nanoparticle detection with IRIS has shown promising results for protein [4] and DNA arrays with attomolar detection sensitivity. …”

Detection and Digital Resolution Counting of Nanoparticles with Optical Resonators and Applications in Biosensing

“…Through the use of an imaging detection approach, the adsorption of analytes upon any region within the field of view may be measured. Imaging-based biodetection utilizing optical sensors has been demonstrated using surface plasmon resonance [22][23][24], photonic crystal (PC) biosensors [25][26][27][28][29], and dielectric thin film interference sensors [30][31][32][33]. In these approaches, NPs, may be detected with the potential to observe the attachment of individual targets.…”

An Interferometric Reflectance Imaging Sensor for Point of Care Viral Diagnostics

“…Further analysis of the detected signal has shown to yield additional information about the size, material, shape, and nanoparticle aspect ratio [17]-[19] to identify the target molecules in the presence of nonspecific binding. The digital detection scheme and orthogonal verification measurements enable a dramatic increase in the sensor’s detection limit compared to traditional ensemble measurement techniques, such as fluorescence, in both serum and whole-blood [20]. In this paper, we present an automated and easy-to-use biosensor imaging platform (see Fig.…”