Fluorescence Sensing of Circulating Diagnostic Biomarkers Using

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Fluorescence Sensing of Circulating Diagnostic Biomarkers Using Molecular Probes and Nanoparticles Oya Tagit, and Niko Hildebrandt ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00625 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Fluorescence Sensing of Circulating Diagnostic Biomarkers Using Molecular Probes and Nanoparticles

Oya Tagita,b* and Niko Hildebrandta* a

NanoBioPhotonics, Center for Nanoscience and Nanotechnology (C2N), Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Université Paris-Sud, CNRS, CEA, 91405 Orsay cedex, France b

Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands. Corresponding Authors Email: [email protected], [email protected]

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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 biomarkers of disease. 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


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Body 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 non-invasive 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 prostate-specific 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 clinically-relevant biomarkers can be found within blood at picomolar or lower concentrations, which corresponds to five to seven 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, identification of multiple biomarkers at a time, so-called multiplexing, provides a better predictive value.


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Table 1. Examples of common circulating tumor markers and their corresponding levels.6-8 Biomarker CA-125

Cancer Type

Sample Analyzed

‘Normal’ LevelsA,B

Ovarian

Blood

< 35 units/mL

Calcitonin

Medullary thyroid

Blood

< 8.5 pg/ mL (men) < 2.5 pM < 5.0 pg/mL (women) < 1.5 pM

AFP

Liver, Testicular

Blood

< 15 units/mL

PSA

Prostate

Blood

< 4.0 ng/ml < 0.12 nM

-hCG

Choriocarcinoma, Germ cell tumors

blood/urine

< 2.5 units/mL (men) < 5.0 units/mL (nonpregnant women) > 25 units/mL (pregnant women)

NMP 22

Bladder

Urine

< 10 units/mL

CTC

Breast, Prostate, Colorectal

Blood

< 1 / 106 – 107 leukocytes

CEA

Breast, Lung, Gastric, Pancreatic, Ovarian, Thyroid, Liver, Melanoma

Blood

< 2.5 ng/mL (nonsmokers) < 14 nM < 5 ng/mL (smokers) < 28 nM

CA-125: cancer antigen 125; AFP: alpha-fetoprotein; PSA: prostate-specific antigen; -hCG: beta- human chorionic gonadotropin; NMP 22: nuclear matrix protein 22; CTC: circulating tumor cells (of epithelial origin); CEA: carcinoembroynic antigen A Molar concentrations were calculated using the following molar masses (with 1 Da = 1 g/mol: M(Calcitonin) = 3.4 kDa; M(PSA) = 34 kDa; M(CEA) = 180 kDa. B units/mL cannot be transferred into molar concentrations.


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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 endpoints 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 early stages.11, 12 Miniaturization of the detection platforms along with increased sensitivity and real-time monitoring ability has made it possible to identify and characterize multiple types of diagnostic biomarkers in complex biological samples.13 Biomarker detection techniques can be classified by their method of signal transduction, which could be photonic, electronic, magnetic etc. Photonic techniques may involve direct detection of the biomolecule of interest or indirect detection of biomolecule-probe conjugates via monitoring the changes in absorbance, luminescence, reflectance, or transmission. The main advantages of photonic techniques involve fast detection, immunity to electrical and magnetic interference, and the ability to provide higher information content.14 The key parameters to evaluate the


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performance of a detection technique include sensitivity, resolution, detection limit, as well as dynamic range, real-time monitoring, and multiplexing capability.15 Broadly defined, sensitivity is the magnitude of a detectable change in the signal in response to changes in the analyte concentration and is determined by the strength of light-matter interactions. Therefore, improvement of these interactions is one of the main concerns for the development of nextgeneration biomolecular detection setups. Reporter molecules with improved photophysical properties, such as high photoluminescence quantum yields and resistance to photobleaching, can increase the sensitivity of a given fluorescence detection technique. Because sensitivity is an indication of signal strength 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


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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.

Biomarker detection using fluorescent labels Among the label-based approaches, methods using fluorescence have found the most widespread application in biomarker detection. Major advantages are the convenience of optical signal transduction routes, the availability of many fluorescent probes with diverse spectral properties, the use of visible light, nondestructive operation, and rapid signal generation and detection. Furthermore, it is possible to achieve very low detection limits and large dynamic ranges using relatively simple instrumentation.26 This section covers the biomarker detection strategies based on fluorescence (or, more precisely, photoluminescence) labeling.

Probes and detection approaches The performance of a particular fluorescence detection system depends largely on the photophysical properties of the probe. Its selection is generally based on chemical and optical 7 ACS Paragon Plus Environment

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suitability, such as stability, spectral output, quantum yield, and cost, for a particular application. Currently, fluorescent probes are available in many forms with a broad range of chemical and photophysical properties. Among the many probes for fluorescence biosensing (Figure 1), organic dyes, such as cyanine dyes (e.g., Cy3 and Cy5), are arguably the most often used. Dyes provide a limited alteration of the conjugated biomolecules, high brightness, and simple labeling procedures.27 Luminescent lanthanide complexes (LC) with extremely long excited-state lifetimes (up to milliseconds) have also found extensive use in biosensing applications.28 LC are composed of a lanthanide cation (usually Tb+3 and Eu+3) that is coordinated by an aromatic ligand, which serves as both a protective shield and a light absorbing antenna.29 The resulting LC provides unique spectral and temporal optical features such as long excited-state lifetimes and multiple, well-separated emission bands in the visible region,30 which render LC promising probes for background-free, multiplexed detection particularly in time-resolved modes.31

The recent breakthroughs in synthesizing metal and semiconductor colloidal nanoparticles (NPs) with unique optical properties have extended the range of fluorescent probes that can be used in label-based biomarker detection. For semiconductor quantum dots (QDs), quantum confinement effects arising from their very small dimensions result in broad absorption spectra in the UVvisible-NIR range, and narrow emission peaks, both of which can be tuned by size, composition, and shape of the QDs.32 The unique, tunable optical properties of QDs make them excellent labels for multiplexed, high-throughput screening. Fluorescent beads are another type of NPs that are obtained by encapsulating fluorescent dyes or QDs within non-toxic and relatively inert polymeric particles. Encapsulation is particularly useful to enhance photostability of fluorescent dyes due to shielding effects33 and to introduce aqueous solubility and reduce toxicity for QDs.34


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Figure 1. In fluorescence-based biomarker detection, biomolecules, such as antibodies (AB), are conjugated to different probes that emit (e.g., dyes, lanthanide complexes - LC, noble metal nanoclusters - NC, fluorescent proteins – FP, or quantum dots - QD), or that quench (e.g., gold nanoparticles – Au-NP) fluorescence. Beads (e.g., microbeads - µ-bead) that incorporate many fluorescent probes can be found in various sizes ranging from some nanometers to several micrometers. An overview of different types of fluorophores for biosensing can be found in reference 35.

Currently, affinity-based methods such as immunoassays and hybridization assays, which rely on the ability of biomolecules (e.g., antibodies or nucleic acids) to specifically recognize and bind to target molecules, are regarded as the reference methods for label-based biomarker detection.36 Biomarker detection in solution can be achieved via mainly four methods (Figure 2). (A) Heterogeneous competitive assay: Capture molecules are immobilized on a surface (e.g., inside the well of a micotiter plate) and then incubated with fluorophore-conjugated biomarkers


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and the solution of interest, which contains the biomarker in an unknown concentration. This incubation leads to a competition between conjugated and free biomarkers for binding to the capture molecules. After incubation, all unbound assay components are separated (washing step) and the fluorescence intensity is measured. Due to the competition between fluorescent and free biomarkers the fluorescence intensity decreases with biomarker concentration. (B) Heterogeneous sandwich assay: Capture molecules are immobilized on a surface and then incubated with the solution of interest, which leads to binding of the biomarkers to the capture molecules. After incubation and washing, a solution containing fluorophore-conjugated capture molecules (which bind to a different epitope of the biomarker) is added. After incubation and washing, the fluorescence intensity is measured. In this case the fluorescence intensity increases with biomarker concentration. (C) Homogeneous competitive assay: Fluorophore-A-conjugated capture molecules are incubated with fluorophore-B-conjugated biomarkers and the solution of interest, which contains the biomarker in an unknown concentration. Washing is not required because a specific energy transfer (ET) signal from fluorophore-A to fluorophore-B is only generated upon binding with fluorophore-conjugated biomarkers. Due to the competition between fluorescent and free biomarkers the fluorescence intensity decreases with biomarker concentration. (D) Homogeneous sandwich assays: Two different capture molecules conjugated with fluorophore A and B, respectively, are incubated with the solution of interest, which contains the biomarker in an unknown concentration. Washing is not required because a specific energy transfer (ET) signal from fluorophore-A to fluorophore-B is only generated upon binding of the biomarker with both fluorophore-conjugated capture molecules. In this case the fluorescence intensity increases with biomarker concentration.


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All four detection schemes come with advantages and disadvantages. In general, sandwich assays are more sensitive and specific.37 However, in the case of small biomarkers that do not provide two different binding sites, competitive assays are inevitable. Heterogeneous assays implemented on biomolecular arrays usually require small amounts of samples and can provide high-throughput detection particularly in microarray format.38 However, often a signal amplification step is necessary for the detection of low-abundance biomolecules. Homogeneous assays have been developed as simpler “mix-and-measure” alternatives without the requirement of washing and separation steps.31 However, the background interference of the non-target molecules in the assay volume can interfere with the detection signal. The following sections give a concise overview of heterogeneous assays on biomolecular arrays, homogeneous assays in solution, and biomarker assays that use nanoparticles.


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Figure 2. Schematic illustration of heterogeneous competitive (A), heterogeneous sandwich (B), homogeneous competitive (C), and homogeneous sandwich (D) fluorescence binding assays. With increasing biomarker concentration, the fluorescence signal (of the red fluorophores) decreases for competitive (E) or increases for sandwich (F) assays. ET refers to energy transfer, which enables binding-specific fluorescence and therefore homogeneous detection.

Heterogeneous assays: Biomarker detection on biomolecular arrays Enzyme-linked immunosorbent assays (ELISA) are one of the most commonly used approaches for identification and quantification of biomarkers on planar surfaces. However, the disadvantages associated with this technique, such as lack of multiplexing capability and long 12 ACS Paragon Plus Environment

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experimental times, limit their use as high-throughput platforms compared to easier and faster experimental preparations that are better suited to high-throughput detection.14 Combining fluorescence labeling with microarray technology has been shown as a useful method for the discovery and detection of potential biomarkers of disease.39,40 Microarrays typically involve hundreds to thousands of capture molecules immobilized on functionalized surfaces, which can be simultaneously studied and analyzed at high-throughput. Therefore, microarray technology plays an important role in protein analysis41 and biomarker identification39 with excellent applicability to clinical detection settings.42, 43 Arrays of antibodies,44 nucleic acid aptamers,45 and peptide aptamers46 have been developed in order to determine concentrations of biomolecules in a variety of biological samples including serum, saliva, tears, tissue lysates, and urine. Furthermore, tissue microarrays that consist of representative samples of normal and disease state tissues have enabled high-throughput biomolecule detection at the DNA, RNA, or protein level.47

Although fluorescence conjugation of biomolecular arrays offers accurate and reproducible detection of high-abundance molecules, often sensitivity has not been sufficient for studying molecules of very low-abundance. To overcome the limited photophysical properties of fluorescent probes, signal amplification methods can be introduced into the biosensors.48

Signal enhancement methods on biomolecular arrays

One of the most commonly used approaches for signal enhancement on biomolecular arrays is label amplification, which relies on increasing the number of signal-producing labels per analyterecognition-event.49 Usually, the detection molecule is labeled with a moiety that has either multivalent characteristics or can be multiplied easily. For the former case, biotin/streptavidin 13 ACS Paragon Plus Environment

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affinity binding is commonly used. An example strategy is illustrated in Figure 3. 50 The amplification occurs after the analyte is captured in a sandwich format by two antibodies (a). The biotinylated detection antibody is recognized by streptavidin that can bind four biotin molecules in total (b). Streptavidin can then capture biotin that is bound to a protein possessing multiple bound biotin moieties (c). The free biotins on the protein surface facilitate further attachment of streptavidin (d). Depending on the number of binding cycles, this amplification strategy can generate a huge number of free biotins per biomarker (e). The uppermost biotin layer was targeted with enzyme-labeled streptavidin molecules and colorimetric detection revealed a 100fold lower limit of detection compared to standard techniques.50 Using similar biotin/streptavidin amplification principles, detection of proteins and cancer cells was achieved at sub-pg/mL levels.51

Figure 3. Schematic illustration of signal enhancement using biotin/streptavidin interactions. a) Formation of a sandwich complex via binding of a biotinylated detection antibody to the biomarker (antigen) on the capture antibody. b) Streptavidin binding to biotin on the detection antibody. c) Introduction of biotinylated protein bearing multiple biotin molecules. d) Formation of another streptavidin/biotinylated protein layer. e) After multiple binding cycles, the final product contains a large number of biotins per antigen. The uppermost biotin layer is eventually 14 ACS Paragon Plus Environment

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targeted with streptavidin-conjugated horseradish peroxidase enzyme, which enables the generation of a calorimetric signal that can be readily measured. Reproduced with permission from reference 50. Copyright 2013 The Royal Society of Chemistry.

Biotin/streptavidin amplification has also been exploited to improve fluorescence detection of biomarkers. Using biotinylated and streptavidin-conjugated quantum dots, both multiplexing (of four different biomarkers) and high sensitivity (subattomolar LODs) could be demonstrated.52 In another work, chemiluminescent immunoassays provided biomolecular detection at subpicogram per milliliter level.53 The flow-through analysis approach that was employed in this work allowed for automation-friendly highly sensitive detection.

Another highly important amplification strategy for signal enhancement is isothermal amplification of nucleic acids.54 Among the various techniques, rolling circle amplification (RCA) is frequently used for the amplification of labels on biomolecular arrays. RCA is an enzymatic process via which long, single stranded DNA molecules (ssDNA) are synthesized on a short, circular ssDNA template using a single DNA primer (Figure 4).49 This highly sensitive and specific amplification tool has found various uses in nucleic acid diagnostics,55 chemiluminescence immunoassays,56 and biosensing.57 The principle of fluorescence signal enhancement using RCA relies on the hybridization of multiple, fluorescently-labeled complementary oligonucleotides to the long RCA product. The presence of multiple fluorescent labels per biomarker allows for the development of highly sensitive assays.


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Figure 4. Schematic of rolling circle amplification (RCA) for biomarker-specific signal enhancement. The detection antibody labeled with an oligonucleotide primer specifically binds to the target (biomarker). A circular DNA acts as the template for the DNA synthesis in the presence of polymerase and nucleotides. The RCA product, which consists of many sequence repeats that are complementary to the DNA template, is detected by hybridization of multiple fluorophore-labeled complementary oligonucleotides. Reproduced with permission from reference 49. Copyright 2016 The Royal Society of Chemistry.

Biotin/streptavidin and RCA amplification can also be combined, as shown for the sensitive detection of human immunoglobulin with a detection limit of 0.9 fM (Figure 5).58 The target was specifically recognized by a biotinylated antibody, and streptavidin binding provided three more biotin binding sites that could capture biotinylated primers for RCA. RCA has also been extended to multiplexed detection. Sensitive, two-color detection of micro-RNA expression in two different cell types was accomplished via priming the RCA with the micro-RNA target


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molecules.59 This intracellular amplification strategy provided information not only on the abundance, but also on the localization of target miRNA molecules.

Figure 5. Principle of a combined biotin/streptavidin-RCA label amplification approach. Biotinylated capture antibodies specifically bind to the target (biomarker) and after streptavidin binding, biotinylated primer DNA and circular DNA templates lead to RCA on the remaining three biotin binding sites of streptavidin. Amplified fluorescence is generated by staining the RCA product with SYBR green fluorescent probes. Reproduced from reference 58. Copyright 2012 The American Chemical Society.

Although label-based techniques for biomolecule detection on planar surfaces provide great levels of sensitivity via signal enhancement, the various experimental steps, such as biomolecule immobilization and enzymatic amplification, present major limitations for a practical use in routine clinical diagnostics. In this respect, biomolecule detection assays in solution that do not require any immobilization or washing/separation steps, and nanoparticle-based assays that can 17 ACS Paragon Plus Environment

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provide sensitivities without the necessity of signal enhancement steps, can be considered as faster, simpler and less expensive alternatives.

Homogeneous assays: Biomarker detection in solution Suspension assays for biomolecule detection are advantageous and more compatible with the complex nature of the biological samples due to faster binding kinetics in solution and elimination of washing (or separation) steps. In addition, these assays can be applied to the detection of various biomarkers without the need to optimize biomolecule immobilization, which make them faster and less expensive than heterogeneous assays.60 In homogeneous assays, the detection can be achieved by probing the polarization,61 intensity,62 or lifetime63 of fluorescence.

Fluorescence polarization assays

In fluorescence polarization assays (FPA), a fluorescent label attached to a capture molecule or an analyte is excited by plane-polarized light. The resulting fluorescence is depolarized due to the rotational motion of the fluorophores in solution (Figure 6). Because the degree of depolarization is proportional to the molecule size, molecular binding events can be assessed by measuring the emitted fluorescence intensity in the vertical and horizontal planes of polarization.64, 65


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Figure 6. Principle of fluorescence polarization assays. When a small fluorescent molecule is excited by plane-polarized light, the resulting emission is depolarized due to rotational motion of the fluorophore in solution. Binding to a large biological target results in a higher degree of emission polarization due to a slower rotation of the fluorophore/biomolecule complex. Reproduced with permission from reference 64. Copyright 2015 The Royal Society of Chemistry.

Several studies have demonstrated the use of FPA for detection of molecules such as toxins,66 drugs,64 peptides,67 and disease biomarkers.68 Duplex detection was also shown for tumor markers by using two-color quantum dots in FPA-based detection schemes, in which clinicallyrelevant detection limits were achieved.61 Although FPA is compatible with automated, highthroughput analysis, it is usually limited to the detection of small molecules and requires more sophisticated instrumentation compared to the standard spectrofluorometers. Furthermore, multiplexing capability has been limited to duplex detection so far. Fluorescence polarization is most often used in research and FPA only found minor application in clinical diagnostics, where 19 ACS Paragon Plus Environment

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other homogeneous detection approaches, such as FRET, provided better suitability to the clinical requirements.

FRET assays Most homogeneous detection assays rely on Fӧrster resonance energy transfer (FRET)-based strategies. FRET has been one of the most prominent techniques to probe biomolecular interactions and molecular conformational changes.69, 70 FRET is a non-radiative process, where energy is transferred from an excited donor molecule (or particle) to an acceptor molecule (or particle) in close proximity.71 The FRET efficiency (EFRET) is proportional to the inverse sixth power of the donor-acceptor distance. It also depends on the extent of spectral overlap between donor emission and acceptor absorption, relative orientation of their transition dipole moments, and quantum yield of the donor.71 The Förster distance (R0) is the donor-acceptor separation distance at which EFRET is 50 %. R0 increases with larger spectral overlap and higher acceptor extinction coefficients. Since FRET occurs over distances at biologically-relevant length scales (~1-20 nm), many biological phenomena that produce changes in molecular proximity (e.g., ligand-receptor binding, molecular structural changes) can be investigated using this technique. In this context, homogeneous FRET assays have been widely used for sensitive detection of molecules such as drugs,72 antigens,62, 73 proteins,74 peptides,75 and cancer biomarkers.76

Despite the simplicity of homogeneous FRET detection, background fluorescence interferences caused by molecules present in the complex biological fluids may mask the detection signal. This problem has been alleviated by the development of time-resolved or time-gated FRET (TRFRET or TG-FRET) analysis. TR-FRET utilizes LC with multiple, well-separated photoluminescence (PL) peaks (Figure 7 a) and PL lifetimes in milliseconds range. When a time 20 ACS Paragon Plus Environment

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delay of 1 to 100 μs is introduced between excitation and PL measurement, non-specific, shortlived background signals can be eliminated (Figure 7 b).

a

b

Figure 7. a) Absorption (black curve) and emission spectra (green curve) of a luminescent terbium complex.31 Copyright 2014 Elsevier B.V. b) Principle of TR-FRET detection. The excited donor with long PL decay time (blue curve) transfers its energy to a near-by acceptor with short PL lifetime (green curve). The sensitized emission of the acceptor (red curve) is measured after a time delay to suppress background, i.e. matrix fluorescence (yellow curve). Adapted with permission from reference 77. Copyright 2014 Nature Publishing Group.

Many studies have reported TR-FRET detection of biomolecules,78, interactions,80,

81

79

biomolecular

and biomolecular conformations77 using LC as FRET donors. Further

advancements in TR-FRET have been achieved using multiple-wavelength fluorophores as acceptors for multiplexed molecular detection.82-84 In this context, several studies have reported the duplexed

63, 85

and triplexed82, 83 detection of molecular binding events using LCs as FRET

donors.


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We have recently extended LC-based multiplexing to the detection of sensitized emission from five acceptor dyes to probe five different lung cancer biomarkers in a single 50 L serum sample (Figure 8).84 The five terbium-dye donor-acceptor combinations provided Förster distances between 4.4 and 6.0 nm. The spectral cross-talk between dyes was efficiently corrected using an inverted matrix acquired in single LC-dye measurements before multiplexing, which allowed for discrimination and quantification of individual markers at concentrations ranging from 0.5-fold to 10-fold of the clinical cut-off values, which are the concentrations at which normal and abnormal biomarker concentrations are distinguished. Very high sensitivity and direct applicability of TR-FRET detection schemes are strong arguments for integration of multiplexed detection into real-life in vitro diagnostics.


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Figure 8. Multiplexed LC-to-dye FRET immunoassay for the simultaneous detection of five lung cancer tumor markers. The top images show the different antibody pairs labeled with terbium complexes (Tb) and five different organic dyes (D1−D5) before (left) and after (right) addition of the tumor markers. The bottom graphs indicate the resulting time-gated PL intensities, which are proportional to the different tumor marker concentrations. Reprinted from reference 84. Copyright 2013 The American Chemical Society.

Although homogeneous TR-FRET assays have been shown to be suitable for highly sensitive multiplexed detection, spectral crosstalk between multiple fluorescent labels remains a challenge. 23 ACS Paragon Plus Environment

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Therefore, a careful choice of fluorescent probes is necessary. Ideally, it should be possible to excite multiple probes using the same source and probes should possess narrow, non-overlapping emission peaks. In this respect, multiplexed detection based on nanoparticles (NPs), in particular semiconductor quantum dots (QDs) holds a great promise toward the development of highly sensitive detection of multiple biomarkers. The following section reviews NP-based detection schemes.

Nanoassays: Biomarker detection using nanoparticles Recent technological advancements in the field of nanotechnology have enabled the development of novel nanoparticle probes with unique properties. Application of these novel nanostructures in biomarker detection schemes has been adapted to both heterogeneous and homogeneous assays. The following sections give a concise overview of nanoparticle-based detection of biomarkers both on planar surfaces and in solution.

Quantum Dots

Since their first applications in a biological context, QDs have gained a great deal of interest as fluorescent labels in biosensing and imaging.86 Many reviews covering biological applications of QDs can be found in the literature.60, 87-89 Among those applications, clinically-relevant ones are in the focus of this review.

Size-tunable optical features of QDs provide high flexibility in the selection of a suitable excitation wavelength and ensure minimal spectral overlap between multiple QD emissions, which make them excellent labels for multiplexed, high-throughput screening.90 Compared to organic dyes, QDs have much larger extinction coefficients and highly reduced photobleaching 24 ACS Paragon Plus Environment

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rates.86 In addition, their large surface area enables conjugation of multiple biomolecules to introduce water solubility, biocompatibility, and further functionalities such as targeting capability. Being considered cumulatively, these unique properties render QDs a powerful tool for biomarker detection. One prerequisite for using QDs in biomarker detection is conjugation of biomolecules with specific recognition capabilities on the QD surface.91, 92 Such QD-antibody conjugates can be either used in the development of immunoassays for detection of biomolecules or they can be used as contrast agents to visualize biomarkers in fixed cells.93

Biomarker detection on planar surfaces

Two important advantages of QD-based biomarker detection include multiplexing and highthroughput screening capabilities. For instance, simultaneous detection of four proteins with four different QDs in a western blot assay,94 and five toxins using five different QDs have been shown.95 A multiplexed assay based on QD labeling was implemented for simultaneous detection of Down’s syndrome biomarkers.96 Two types of capture antibodies that specifically recognized the target analytes α-fetal protein (AFP) and β-human chorionic gonadotrophin (HCG) were immobilized on a membrane (Figure 9 I a). A sandwich was formed via binding of QD-labeled detection antibodies to the captured biomarkers (Figure 9 I b). The limits of detection obtained for AFP and HCG were as low as 1 ng/mL and 1x10-6 IU/L, respectively. Advanced nanofabrication techniques can produce functional surfaces to be used in QD-based detection schemes. For instance, QD-based micro- and nanoarrays were fabricated using e-beam lithography.97 The arrays were used for the detection of proteins, gold nanoparticles (AuNPs), and magnetic microparticles via biotin/streptavidin affinity binding (Figure 9 II). Furthermore, cellular adhesion, spreading, and migration studies performed on these arrays revealed


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information on cellular dynamics. Another example demonstrating the functional surfaces for QD-based biomarker detection was developed as a lateral flow test strip (Figure 9 III).98 The detection principle was divided in i) binding of antibody-functionalized QDs to antigens in the sample solution, ii) lateral flow of the mixture on the test strip via capillary forces, iii) capture of the QD-antibody-antigen complexes by immobilized antibodies in a detection zone toward the end of the test strip, and iv) measurement of the QD fluorescence intensity to quantify the antigen concentration. Nitrated ceruloplasmin spiked into human plasma samples could be quantified with a detection limit of 8 ng/mL, with a test duration of only 10 minutes. Such simple lateral flow test strips have the potential to be integrated in consumer diagnostics (similar to pregnancy tests).


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Figure 9. I) Schematic of a duplexed immunoassay using green and red emitting QDs. Two types of capture antibodies specific for two different biomarkers are immobilized on a substrate (a). Detection antibodies labeled with green and red QDs form a sandwich structure upon binding to biomarkers captured on the surface (b). Reproduced with permission from reference 96. Copyright 2013 Elsevier B.V. II) Application of QD micro- and nanoarrays produced by ebeam lithography for the investigation of proteins (left), AuNPs (center), and cellular dynamics (right). Reproduced from reference 97. Copyright 2013 The American Chemical Society. III) Fluorescence images of QD-based lateral-flow test strips for the detection of nitrated ceruloplasmin (QD fluorescence detection zone in red) at concentrations of 10 µg/mL (A), 1 µg/mL (B), 100 ng/mL (C), 10 ng/mL (D), and 0 ng/mL (E - control). Reproduced from reference 98. Copyright 2010 The American Chemical Society.

In another approach, in which QDs were used for triplexed detection of lung cancer biomarkers, micron-size magnetic particles were used as immune carriers.99 This inexpensive and simple method required only 20 L sample volume and achieved simultaneous detection of three markers down to 1 ng/mL. Doping of different amounts of QDs with different colors inside microbeads has been used for the creation of optical barcodes.100 The combination of six colors with ten different intensities would theoretically result in one million different optical codes and the conjugation of such beads to a large variety of biomarkers would enable high-throughput screening of biomolecules.90 For instance, a recent work reported the development of QDencoded microbead arrays within microfluidic devices for multiplex immunoassays. The microbead-on-chip assay was used for simultaneous detection of prostate-specific antigen and albumin.101 In a more clinical context, a full validation of QD barcode technology for Hepatitis B diagnosis has been reported.87 The detection of multiple regions of the viral genome using multiplexed QD barcodes was shown to improve clinical sensitivity from 54.9–66.7% to 80.4– 90.5%. Construction of encoded-bead libraries for the identification of biomarkers would provide 27 ACS Paragon Plus Environment

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fast and simple detection platforms for multiplexed, high-throughput screening in clinical applications.

FRET assays

The ability to excite QDs at almost any wavelength below their emission peaks, their large absorption cross section over a broad spectral range, and their size- and material-tunable absorption and emission wavelengths, make QDs excellent FRET donors and acceptors.87 FRETbased analyses of proteins,102 nucleic acids103 as well as other biomolecules69 have been reported using QDs. Since the FRET efficiency depends on the D-A separation distance, the main limiting factor for utilization of QDs in FRET-based detection is their relatively large size, which is the cumulative result of the core/shell inorganic QD, the organic coating for aqueous solubility, and the attached biomolecules. In particular, sandwich immunoassays, which require two relatively large antibodies, are challenging systems. An approach to rise to these challenges is the combination of terbium-to-QD FRET, which can provide large R0 values and reduced background due to time-gated luminescence detection, and versatile antibody conjugation strategies.63,

104-107

In particular, the use of smaller antibodies (F(ab) or F(ab)2 fragments and

VHH nanobodies),63,

104, 106, 107

oriented conjugation,104,

108

conjugation via reduced cysteines of antibodies using direct or

and smaller biocompatible QD-coatings105 could improve the

sensitivity of such homogeneous QD-based immunoassays. Alpha-fetoprotein (AFP),109 carcinoembryonic antigen (CEA),110, 111 the epidermal growth factors EGFR and HER2 (even in a duplexed format),104, 107 and prostate specific antigen (PSA)63, 106, 108 have been quantified at clinically relevant concentration ranges. We recently extended terbium-to-QD FRET assays to the detection of short nucleotide sequences.112 A homogeneous and triplexed microRNA


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detection assay provided detection limits of 0.2 nM for triplexed and 0.06 nM for single microRNA detection. These studies demonstrated the feasibility of integrating QD-based homogeneous assays into multiplexed diagnostics for the detection of biomarkers of disease.

Other Nanoparticles

In addition to QDs, metal and silica NPs have been extensively applied as fluorescent probes for biosensing. Although noble metal NPs do not fluoresce, unless they become extremely small and are then usually referred to as nanoclusters,113, 114 they are known to display collective oscillation of free electrons, which results in intense absorption peaks in the visible region, which can be exploited to quench the fluorescence of appropriate energy donors. Most often AuNPs are applied in such fluorescence-quenching biosensors.115-117 Furthermore, interactions between the surface plasmon field of metallic NPs and dipole moments of near-by fluorophores can result in fluorescence enhancement, which is called metal-enhanced fluorescence (MEF).118 The MEF concept has been used for improvement of biomolecular detection sensitivity on functional microarray surfaces as well as solution-based assays.119, 120 For instance, multiplexed detection of platelet-derived growth factor (PDGF-BB) and thrombin was achieved at picomolar concentrations (Figure 10).120 The aptamer-modified, biotinylated AgNPs immobilized on streptavidin microarrays acted as capture substrate to specifically bind target proteins. After specific aptamer-protein binding, a classical sandwich structure was formed upon specific binding of fluorescently-labeled aptamers. This sandwich assay based on dual aptamers and AgNPs resulted in a detection limit of 625 pM for PDGF-BB and 21 pM for thrombin.


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Figure 10. Illustration of the aptamer-based sandwich assay process for one-spot multiplex detection of thrombin and PDGF-BB. Aptamer-modified, biotinylated AgNPs were immobilized on streptavidin patterns on a glass slide. The target proteins were captured by specific binding of aptamers. Fluorescently-labeled aptamers were bound to proteins forming a sandwich pattern. Different emission wavelength of fluorescent tags on aptamers enabled simultaneous detection of two different proteins. Reproduced with permission from reference 120. Copyright 2016 Elsevier B.V.

Along with enhancement of fluorescence for biomolecule detection,121 AgNPs have also been reported to enhance the FRET efficiency.122 An AgNP-enhanced FRET sensing system was designed for the sensitive detection of human platelet-derived growth factor-BB (PDGF-BB) with the detection limit of 0.8 ng/mL.122 In a recent study, silver triangular nanoparticles (STNPs) were designed as FRET acceptors in homogeneous antigen detection assays, in which QDs acted as donors.123 The absorption spectra of tailor-designed STNPs matched very well with QD emission peak for improved FRET efficiency. The detection sensitivity was found to be 27 30 ACS Paragon Plus Environment

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times higher compared to other QD/metal NP FRET pairs. This tailor-design approach to maximize the spectral overlap can be adjusted for different QDs to optimize metal-enhanced FRET over long distances. Although the exact type of energy transfer is still under debate, the longer distances of energy transfer to metal NPs (compared to classical dipole-dipole FRET) has mainly been related to the large surfaces of these NPs, which leads to a r-4 (instead of r-6) distance dependence (nanosurface energy transfer – NSET).124

Silica nanoparticles (SiNPs) doped with fluorescent dyes have also found extensive use in biomolecular detection applications.125 Encapsulating fluorescent dyes within non-toxic and relatively inert SiNPs does not only improve the signal intensity, but also the photostability of the fluorescent dyes due to shielding effects.33 In addition, encapsulation of QDs within SiNPs has proven useful to introduce aqueous solubility and reduce toxicity while maintaining the unique optical properties of QDs.34 Moreover, co-encapsulation of QDs and magnetic NPs within SiNPs has enabled the development of multimodal contrast agents.34 Bimodal, magneticallyencoded fluorescent SiNPs ((CdTe/Fe3O4)@SiO2) were shown to achieve magnetic separation, capture, and fluorescent detection of three antigens (cancer antigen 125, AFP, and CEA) with detection limits of 20 kU/L, 10 ng/mL, and 5 ng/mL, respectively.34 This strategy was proposed to be applicable to multicomponent separation and analysis of biomolecules in a facile, rapid, and economical way. In addition to encapsulation strategies, immobilization of QDs on SiNP surfaces has allowed for the formation of nanocomposite probes.126 A lateral-flow test strip was designed using QD/SiNPs as label probes. Detection of a model protein was reported to be 10 times more sensitive compared to conventional test strips.


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Another class of nano- (and micro-) particles that have found widespread use in biomarker research is based on dye-doped polystyrene beads, which are commercially-available in a number of different sizes and colors. For instance, the AlphaScreen (amplified luminescent proximity homogeneous assay screen) technology platform from Perkin Elmer was developed from a diagnostic assay known as luminescent oxygen channeling immunoassay (LOCI). The assay comprises two polystyrene beads (approximately 200 nm in diameter), designated as donor (D) and acceptor (A), which form pairs in the presence of an analyte (Figure 11).127 Donor beads are doped with a photosensitizer that generates singlet oxygen when excited at 680 nm. The singlet oxygen can propagate through the solution and react with the acceptor beads to produce a chemiluminescent signal. Initial detection schemes of AlphaScreen assays involved the use of acceptor beads containing three chemical dyes with a spectral output at 520-620 nm. However, today also acceptor beads with europium and terbium luminescence (mainly at 615 nm and 545 nm, respectively) are available.

Figure 11. AlphaScreen assay principle. When the donor (D) is excited at 680 nm, ambient oxygen is converted to singlet oxygen. Singlet oxygen molecules that propagate to the acceptor bead (A) can trigger chemiluminescence. Adapted with permission from reference 127.


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In order to produce a detectable signal, donor and acceptor beads need to be bound to each other, which is usually achieved via an immunological complex. Reactive bead surfaces enable conjugation of many different ligand-receptor partners. In theory, AlphaScreen beads can be separated as far as 200 nm, but in practice the system needs to be carefully adapted to the selected biological interaction to be analyzed. In the past decade, AlphaScreen technology has been used for studying protein-protein interactions,128,

129

protein-nucleic acid interactions,130

high-throughput aptamer identification,127 rapid detection and measurement of insulin,131 highthroughput screening of neurotoxic oligomers,132 as well as identification of inhibitor molecules in drug discovery.133 One recent study reported a direct comparison of AlphaScreen and ELISA assays for the detection of insulin secreted from stimulated cells.131 Although LOD values obtained for both methods were similar, a ~ 50 % improvement in signal-to-noise ratio was achieved for the AlphaScreen assay. Although the homogeneous nature of AlphaScreen makes it an attractive technology for probing a variety of molecular interactions, the technology is not adaptable to all fluorescence readers and thus more limited compared to other luminescence assays.

Conclusion and Future Perspectives Significant opportunities that exist at the interface of optical techniques, nanotechnology, and molecular diagnostics have enabled the development of next-generation fluorescent probes and detection techniques. The advantages offered by these techniques have successfully been coupled to the rapidly-expanding field of biomarker detection and discovery. In addition, advancements in miniaturization of the detection platforms have proven useful for integration of various detection schemes for high-throughput and multiplexed screening of complex biological 33 ACS Paragon Plus Environment

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samples. Furthermore, utilization of nanoparticles with unique optical properties not only introduced superior detection sensitivity but also minimized the sample requirement. Despite the immense progress that has been achieved, detection techniques are still facing several biological and technological issues that need to be resolved. For instance, retaining the activity of biomolecules during conjugation to fluorescent probes and improving the detection limits and sensitivity, particularly via suppressing non-specific background signal originating from the complex nature of biological samples, remain challenging. Background suppression and higher order multiplexing can be achieved by using novel fluorophores and fluorophore combinations, including lanthanide complexes, quantum dots, or gold nanoparticles. On the basis of the recent advances in detection techniques, we anticipate significant improvements in the near future toward the generation of robust, sensitive, and reliable platforms for early detection of diagnostic biomarkers.

Glossary Biomarker: According to the National Institutes of Health Biomarkers Definitions Working Group, a biomarker is defined as ‘a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.’ In this review we refer to circulating biomarkers, which are biological molecules (such as proteins or nucleic acids) found in biological fluids (so-called liquid biopsies) that are specifically linked to particular disease states. Sensitivity: Magnitude of a detectable change in the signal in response to changes in the measured quantity. Resolution: Smallest magnitude of change that can be detected in the measured quantity.


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Limit of detection (LOD): The minimum resolvable signal at a given noise level. Multiplexing: Detection of multiple signals that correspond to multiple measured quantities from a single sample volume. Fӧrster resonance energy transfer (FRET): A non-radiative transfer of energy from an excited donor to an acceptor in close proximity. Quantum dots (QDs): Luminescent semiconductor nanoparticles (circa 2 – 10 nm in diameter) with unique optical properties that can be tuned by their size.

Acknowledgements Financial support by European Commission EU 7th framework Programme (grant agreement n°246556) is acknowledged.

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TOC Figure. The detection of biomarkers circulating in body fluids is of utmost importance for the diagnosis and therapy of diseases. Detection technologies using fluorescent probe-based approaches, with fluorophores ranging from small organic dyes, over nanoparticles, to microbeads, play a major role in the quantification of circulating biomarkers in complex biological samples. This review explains the most common techniques and discusses representative applications from the recent literature.


Fluorescence Sensing of Circulating Diagnostic Biomarkers Using

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