Nanoparticle-enhanced Imaging Based Plasmonic Biosensor

Efficient medical care fundamentally relies on the ability to provide a timely and accurate diagnosis. Thanks to advances in biomedical research, specific molecules called diagnostic molecular biomarkers have been discovered in the human body that help indicate diseases in highly specific ways. The small size and low concentration of many of these molecules pose a serious challenge to detecting them from the rich content of human blood and other liquid biopsies. Medical laboratories use large and complex tools to enable sensitive and robust quantification of such biomarkers. However, these approaches are time-consuming, require expensive equipment and delay the doctors' decision-making. Therefore, compact, cost-effective and rapid technologies that enable testing of biological fluids to identify low-abundance biomarkers directly at the patientâs bedside are critically needed to assist the modern healthcare. This doctoral thesis presents a novel biosensor that enables highly sensitive, accurate and rapid detection of disease biomarkers in a low-cost and portable device directly from patient blood serum. The first major and original contribution is on the introduction of an innovative sensing principle that uses sub-wavelength gold nanoparticles and large area periodic gold nanohole arrays. The nanohole arrays consist of millions of nano-perforations in a thin gold metal film on a glass substrate and enable a plasmonic phenomenon called extraordinary optical transmission. The interactions between nanoparticles and nanoholes are imaged in a spectrometer-free set-up and enable the detection of individual molecule binding in complex samples. Unlike conventional plasmonic sensing approaches that rely on spectral shifts of plasmonic resonances, our method exploits intensity modulations caused by individual nanoparticles on nanohole arrays. Therefore, the technology overcomes classical plasmonic detection limits imposed by refractive index sensitivity. The work shows that the biosensor achieves highly sensitive detection, meeting clinically relevant concentrations, and can provide a powerful platform for biomarkers testing. The second major and original contribution includes the integration of the novel plasmonic sensor technology into a portable point-of-care (POC) device. It is deployed in a hospital and validated with a wide range of patient samples with inflammatory conditions. The device enables ultra-sensitive detection of two sepsis-related biomarkers, procalcitonin, and C-reactive protein. The tests with biobank patient samples revealed that the novel POC device provides diagnostic performance equivalent to gold standard laboratory immunoassays. Moreover, identification of biomarker levels can be performed in under 15 minutes on-site, providing critical advantage compared to laboratory testing. The results of this thesis build upon a broad interdisciplinary knowledge ranging from engineering (including plasmonics, imaging, nanofabrication, and device integration) to chemistry, biology, and medical diagnostics. The plasmonic sensing principle introduced in this work offers a promising strategy for the development of many new biosensing applications, while the developed point-of-care biosensor has the potential to provide a rapid and accurate tool to assist the diagnosis and management of diseases in various settings, improving the quality of medical care for more people.

Rapid and Digital Detection of Inflammatory Biomarkers Enabled by a Novel Portable Nanoplasmonic Imager

Hatice Altug, Alexander Belushkin, Filiz Yesilköy

New point-of-care diagnostic devices are urgently needed for rapid and accurate diagnosis, particularly in the management of life-threatening infections and sepsis, where immediate treatment is key. Sepsis is a critical condition caused by systemic response to infection, with chances of survival drastically decreasing every hour. A novel portable biosensor based on nanoparticle-enhanced digital plasmonic imaging is reported for rapid and sensitive detection of two sepsis-related inflammatory biomarkers, procalcitonin (PCT) and C-reactive protein (CRP) directly from blood serum. The device achieves outstanding limit of detection of 21.3 pg mL(-1) for PCT and 36 pg mL(-1) for CRP, and dynamic range of at least three orders of magnitude. The portable device is deployed at Vall d'Hebron University Hospital in Spain and tested with a wide range of patient samples with sepsis, noninfectious systemic inflammatory response syndrome (SIRS), and healthy subjects. The results are validated against ultimate clinical diagnosis and currently used immunoassays, and show that the device provides accurate and robust performance equivalent to gold-standard laboratory tests. Importantly, the plasmonic imager can enable identification of PCT levels typical of sepsis and SIRS patients in less than 15 min. The compact and low-cost device is a promising solution for assisting rapid and accurate on-site sepsis diagnosis.

WILEY-V C H VERLAG GMBH2019

Nanoparticle-Enhanced Plasmonic Biosensor for Digital Biomarker Detection in a Microarray

Hatice Altug, Alexander Belushkin, Filiz Yesilköy

Nanoplasmonic devices have become a paradigm for biomolecular detection enabled by enhanced light–matter interactions in the fields from biological and pharmaceutical research to medical diagnostics and global health. In this work, we present a bright-field imaging plasmonic biosensor that allows visualization of single subwavelength gold nanoparticles (NPs) on large-area gold nanohole arrays (Au-NHAs). The sensor generates image heatmaps that reveal the locations of single NPs as high-contrast spikes, enabling the detection of individual NP-labeled molecules. We implemented the proposed method in a sandwich immunoassay for the detection of biotinylated bovine serum albumin (bBSA) and human C-reactive protein (CRP), a clinical biomarker of acute inflammatory diseases. Our method can detect 10 pg/mL of bBSA and 27 pg/mL CRP in 2 h, which is at least 4 orders of magnitude lower than the clinically relevant concentrations. Our sensitive and rapid detection approach paired with the robust large-area plasmonic sensor chips, which are fabricated using scalable and low-cost manufacturing, provides a powerful platform for multiplexed biomarker detection in various settings.

2018

Early sepsis diagnosis via protein and miRNA biomarkers using a novel point-of-care photonic biosensor

Hatice Altug, Alexander Belushkin

y Sepsis is a condition characterized by a severe stage of blood-infection often leading to tissue damage, organ failure and finally death. Fast diagnosis and identification of the sepsis stage (sepsis, severe sepsis or septic shock) is critical for the patient's evolution and could help in defining the most adequate treatment in order to reduce its mortality. The combined detection of several biomarkers in a timely, specific and simultaneous way could ensure a more accurate diagnosis. We have designed a new optical point-of-care (POC) device based on a phase-sensitive interferometric biosensor with a label-free microarray configuration for potential high-throughput evaluation of specific sepsis biomarkers. The sensor chip, which relies on the use of metallic nanostructures, provides versatility in terms of biofunctionalization, allowing the efficient immobilization of different kind of receptors such as antibodies or oligonucleotides. We have focused on two structurally different types of biomarkers: proteins, including C-reactive protein (CRP) and Interleukin 6 (IL6), and miRNAs, using miRNA-16 as an example. Limits of Detection (LoD) of 18 mu g mL(-1), 88 mu g mL(-1) and 1 mM (6 mu g mL(-1)) have been respectively obtained for CRP, IL6 and miRNA-16 in individual assays, with high accuracy and reproducibility. The multiplexing capabilities have also been assessed with the simultaneous analysis of both protein biomarkers. (C) 2019 Elsevier B.V. All rights reserved.

ELSEVIER SCIENCE BV2019