Review pubs.acs.org/acssensors
Fluorescence Sensing of Circulating Diagnostic Biomarkers Using Molecular Probes and Nanoparticles Oya Tagit*,†,‡ and Niko Hildebrandt*,† †
NanoBioPhotonics (nanofret.com), Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Université Paris-Sud, CNRS, CEA, 91405 Orsay, France ‡ Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands ABSTRACT: The interplay of photonics, nanotechnology, and biochemistry has significantly improved the identification and characterization of multiple types of biomarkers by optical biosensors. Great achievements in fluorescence-based technologies have been realized, for example, by the advancement of multiplexing techniques or the introduction of nanoparticles to biochemical and clinical research. This review presents a concise overview of recent advances in fluorescence sensing techniques for the detection of circulating disease biomarkers. Detection principles of representative approaches, including fluorescence detection using molecular fluorophores, quantum dots, and metallic and silica nanoparticles, are explained and illustrated by pertinent examples from the recent literature. Advanced detection technologies and material development play a major role in modern biosensing and consistently provide significant improvements toward robust, sensitive, and versatile platforms for early detection of circulating diagnostic biomarkers. KEYWORDS: biosensing, fluorescence, FRET, quantum dots, nanoparticles, clinical diagnostics, immunoassays ody fluids such as serum, urea, and saliva are complex environments that contain a dynamic range of biomolecules, electrolytes, dissolved gases, and waste products, which vary in abundance under certain pathological and physiological conditions. Therefore, these body fluids serve as excellent archives of information regarding the health state of individuals as well as noninvasive means for biological specimen collection.1 Circulating diagnostic biomarkers are biological molecules such as proteins, nucleic acids, cells, and cell lysates that can be found in body fluids. They are specifically linked to certain physiological (e.g., pregnancy) or disease (e.g., cancer) states, which can be clinically monitored using biomarker detection techniques.2,3 These techniques usually involve detection of individual biomolecules (e.g., human chorionic gonadotropin, hCG, in the case of pregnancy and prostatespecific antigen, PSA, for prostate cancer) in body fluids via specific and selective affinity interactions.4 Many different circulating disease markers, such as tumor markers, have been characterized and are in clinical practice (Table 1). Most of the clinically relevant biomarkers can be found within blood at picomolar or lower concentrations, which corresponds to 5 to 7 orders of magnitude lower concentrations than the most abundant plasma proteins.5 Relatively low abundance of disease markers and the complex biological environment render detection of circulating biomarkers challenging. Furthermore, as also shown in Table 1, certain biomarkers can be associated with more than one pathological condition. Therefore,
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© 2016 American Chemical Society
identification of multiple biomarkers at a time, so-called multiplexing, provides a better predictive value. Although FDA-approved biomarker detection tools are already in clinical practice (e.g., FIRST RESPONSE as an indicator of pregnancy, and Access Hybritech p2PSA for diagnosis of prostate cancer), detection of biomarkers of extremely low abundance remains a challenge. For instance, circulating tumor cells can be as rare as 1 per 106−107 leukocytes.7 Furthermore, lack of measurable clinical end points for certain rare genetic diseases render the biomarkers vitally important as surrogates.9 Therefore, the ability to identify disease biomarkers significantly improves diagnosis and monitoring as well as evaluation of the relevant therapeutic strategies.10 The key challenge in diagnosis is to identify multiple, low-abundance biomarkers in complex biological samples with high precision in a time- and cost-efficient way. Therefore, development of novel techniques for simultaneous measurements of multiple clinical parameters from a single volume of complex media is required, which would eliminate the need for several sequential analysis steps related to “traditional” approaches for biomarker detection. Recent technological advances (e.g., in nanotechnology) have enabled the development of next-generation techniques, which serve as useful platforms for the discovery of markers of disease at its Received: October 9, 2016 Accepted: December 6, 2016 Published: December 6, 2016 31
DOI: 10.1021/acssensors.6b00625 ACS Sens. 2017, 2, 31−45
Review
ACS Sensors
only, it cannot be used alone to evaluate the system performance. The limit of detection (LOD) is a direct indicator of system performance and is defined as the minimum resolvable signal at a given noise level. LOD is usually expressed in terms of minimum sample concentration that can be detected (units usually in g/L or molarity). Low LOD values (picomolar or lower) are usually the main target for clinical detection because most of the clinically relevant biomarkers that exist within blood are at picomolar or lower concentrations. Low LODs can be achieved by increasing the signal-to-noise ratio. Nanotechnology has afforded many innovative and flexible approaches that can possibly push the limits of sensitivity and specificity of detection in addition to introducing multiplexing capability and reducing measurement complexity and cost.11 Potential applications of such novel approaches are mainly based on the measurement of an inherent property (e.g., mass, dielectric property) of the biomarker itself (label-free detection)16 or the detection of physical (optical, electronic, magnetic) or chemical (e.g., catalytic) characteristics of biomarker−probe conjugates (label-based detection). 17 Although the focus of this review is fluorescent label-based detection methods, it is noteworthy that several label-free optical techniques also exist to detect circulating biomarkers. One example are surface plasmon resonance (SPR) SPR-based techniques,18 including SPR imaging (SPRi),19 as well as waveguide techniques such as optical ring resonators.20 Applications range from quantitative immunoassays to detect disease biomarkers from human plasma using SPR,21,22 over simultaneous monitoring of multiple biomolecular interactions using SPRi,23 to the detection of cancer biomarkers in serum at clinically relevant concentration ranges24 also in a multiplexed manner25 using optical ring resonators. In the following sections several promising approaches based on fluorescence detection are reviewed. The detection principles and relevant applications of different techniques are discussed in terms of sensitivity and LOD.
Table 1. Examples of Common Circulating Tumor Markers and Their Corresponding Levels6−8 biomarkera
cancer type
sample analyzed
CA-125 Calcitonin
ovarian medullary thyroid
blood blood
AFP PSA
liver, testicular prostate
blood blood
β-hCG
choriocarcinoma, germ cell tumors
blood/ urine
NMP 22 CTC
bladder breast, prostate, colorectal
urine blood
CEA
breast, lung, gastric, pancreatic, ovarian, thyroid, liver, melanoma
blood
“normal” levelsb,c
