Chemistry, Biology, and Medicine of Fluorescent Nanomaterials and

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Chemistry, Biology, and Medicine of Fluorescent Nanomaterials and Related Systems: New Insights into Biosensing, Bioimaging, Genomics, Diagnostics, and Therapy Jun Yao,† Mei Yang,† and Yixiang Duan*,†,‡ †

Research Center of Analytical Instrumentation, Analytical and Testing Center, College of Chemistry, Sichuan University, Chengdu, Sichuan 610064, China ‡ Research Center of Analytical Instrumentation, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, China 7.6. Multifunctional Nanoparticles for Bioimaging 8. Other Fluorescence-Based Applications 8.1. Fluorescence Lifetime Imaging Microscopy 8.2. Fluorescence Polarization Correlation Measurement 9. Conclusion and Perspectives Author Information Corresponding Author Notes Biographies Acknowledgments Abbreviations References

CONTENTS 1. 2. 3. 4.

Overview Introduction Theory and Background Several Classes of Significant Nanoparticles 4.1. Quantum Dots 4.2. Noble Metal Nanoparticles 4.3. Silicon Nanomaterials 4.4. Upconversion Nanomaterials 4.5. Multifunctional Nanoparticles 5. Bioanalysis Applications 5.1. Biological Sensing Technology 5.1.1. FRET-Based Biosensors 5.1.2. Photoinduced Electron Transfer and Charge Transfer 5.1.3. Metal-Enhanced Fluorescence 5.1.4. Multiplexed Assays 5.2. Genomics 5.2.1. Aptamer 5.2.2. Single Nucleotide Polymorphism 5.2.3. RNA Interference 5.2.4. Fluorescence in Situ Hybridization 6. Biomedical Applications 6.1. Pathogen Detection 6.2. Antibacterial Activity 6.3. Immunoassay 6.4. Drug Delivery 6.5. Protein Corona 6.6. Biotherapeutics 6.6.1. Photodynamic Therapy 6.6.2. Hyperthermia 7. Bioimaging 7.1. Near-Infrared Fluorescence Imaging 7.2. Quantum Dots for Bioimaging 7.3. Upconversion Nanoparticles for Bioimaging 7.4. Silica Nanoparticles for Bioimaging 7.5. Carbon Nanomaterials for Bioimaging © XXXX American Chemical Society

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1. OVERVIEW The combination of fluorescence and nanomaterials has developed into an emerging research area: fluorescent nanoparticles. Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology and have attracted increasing interest for bioanalytical labeling applications in recent years. Their unique size-dependent properties make these materials superior and indispensable in many areas of human activity. Meanwhile, fluorescence-based detection is the most common method utilized in biosensing because of its high sensitivity, simplicity, and diversity. The emerging development and innovation of luminescent nanoparticles (NPs) with unique optical properties, yet complicated surface chemistry, paves new roads for fluorescence imaging and sensing as well as for in vitro and in vivo labeling in cells, tissues, and organisms. As a result, this approach is widely employed in various areas of biology and medicine, including proteomic and genomic studies, disease diagnostics, pharmaceutical screening, drug delivery, assembled molecular control, protein purification, biological therapeutics, and medical imaging (such as in vivo imaging, sensing in cancer research, and selective tumor targeting). Specifically, we selected size- and shape-dependent photoluminescence (PL) of quantum dots (QDs) as well as plasmon of metal NPs and discuss their biological applications. This review summarizes the applications of different NPs (including QDs, rare earth doped NPs, gold NPs, or silica NPs) in biosensing and imaging using detection techniques such as

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Figure 1. General steps involved in the application of functional NPs. Reprinted with permission from ref 34. Copyright 2010 American Chemical Society.

fluorescence, fluorescence resonance energy transfer (FRET), fluorescence lifetime measurement, and multiphoton microscopy. Herein, we try to illustrate the main limitations and future directions of NPs in these areas of study.

understanding how these synthetic materials interact with biomolecules,30 cells,31 tissues,32 and ultimately patients33 is central to the application of these systems in biomedicine. To utilize functional NPs for biosensing and bioimaging, one has to focus on the following steps: (a) synthesis, (b) coating, (c) surface functionalization or bioconjugation, and (d) applications, as depicted in Figure 1.34 Among these steps, functionalization (surface modification) is required to make the NPs more soluble and stable in aqueous media (hydrophilicity). This increases biocompatibility and biofunctionality while preserving their original properties,35 which are vital for biomedical applications (Figure 2).36

2. INTRODUCTION The investigation of many fundamental processes in life sciences requires straightforward tools for fast, sensitive, reliable, and reproducible detection of biomolecular interaction among various molecular or ionic species. One of the best suited and most popular methods to meet these challenges is the use of photoluminescence or fluorescence techniques in conjunction with functional dyes and labels.1−3 Sensitive detection of target analytes present at trace levels in biological samples often requires labeling of reporter molecules with fluorescent dyes. Fluorescence detection is by far the dominant detection method in the field of sensing technology due to several well-established advantages, including the following.4 (a) Rapid signal acquisition that each individual fluorescent label can offer approximate 107−108 photons per second for determination. (b) Multi fluorescence dyes can be used for multiplexed assays. (c) High detection sensitivity (such as single molecule detection). (d) The luminescent signal is localized (different from some enzyme-linked based amplification strategies). (e) The labeling procedure can be straightforward provided that suitable functional groups are available on the target analyte. However, it can be difficult to reach a low detection limit in fluorescence detection due to the limited extinction coefficients or quantum yields of traditional organic dyes and also low dye-to-reporter molecule labeling ratio. The recent explosion of nanotechnology, leading to the development of materials with submicrometer-sized dimensions and unique optical properties, has opened up new horizons for fluorescence detection. Nanomaterials can be made from both inorganic and organic materials and are less than 100 nm in length along at least one dimension. The development of functional NPs has progressed exponentially over the past two decades. NPs are attracting considerable interest as viable biomedical materials, and research in nanotechnology is growing due to their unique physical and chemical properties. Examples of the diverse range of available NPs include: noble metal NPs (e.g., Au,5 Ag,6,7 Pt,8 and Pd9), semiconductor nanocrystals (e.g., CdSe, CdS, ZnS,5,10 TiO2,11 PbS,12 InP,12 and Si13), magnetic compounds (e.g., Fe3O4,14 CoFe2O4,15 FePt,9 and CoPt16), upconverting nanocrystals,17 silica NPs,18 carbon NPs,19 and their combinations (core−shell NPs and other composite nanostructures). NPs provide promising platforms for a wide variety of biomedical applications, including biosensing,20−22 bioimaging,23−25 drug delivery26−28 and gene therapy.29 Clearly,

Figure 2. Scheme of the most relevant strategies to modify the surface of the NPs: (above) to render NPs solubility in aqueous environments; (below) to address biological functionality to the NPs. Reprinted with permission from ref 36. Copyright 2008 Elsevier.

Also, most of the applications described above rely on fluorescence spectroscopy, one of the most informative and sensitive analytical techniques that has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells, and organisms relies on the development of fluorescence-based tools. Particularly, as is going to be described below, different fluorescence techniques have been widely employed in such developments, including conventional fluorescence spectroscopy, fluorescence correlation spectroscopy (FCS), time-resolved fluorescence spectroscopy, steady-state fluorescence, near-infrared (NIR) fluorescence and fluorescence microscopy. The aim of this review is, first, to give reader a historic prospective of nanomaterial application to biology and medicine, and second, to comment the recent advances in this particular field. At the same time, we will provide an B

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introduction to measurements and methods associated with the fluorescence spectroscopy. Additionally, the necessary theoretical background to understand the general and most common processes affecting the fluorescence is also provided. The focus of this review is to highlight developments in the use of fluorescent NPs for bioanalytical and optical imaging applications, especially in the field of proteomic and genomic studies (e.g., bioaffinity sensors for detection of nucleic acids and proteins). In addition, inorganic NP labels based on noble metals and QDs, combined into highly selective recognition compounds (e.g., antibodies or more recently aptamers) appear to be the most versatile systems and are discussed eminently. Lastly, we will briefly mention some modern instrumental techniques for fluorescence analysis. Readers interested in detailed information are directed to a number of excellent reviews and books that cover these topics.37−44

Figure 3. Jablonski diagram and a time scale of photophysical processes. Reprinted with permission from ref 48. Copyright 2010 American Chemical Society.

Spain called Lignum nephriticum used as a medical treatment for liver and kidney ailments.50−52 The magical optical phenomenon was later investigated by Kircher, Grimaldi, Boyle, Newton, and many other scientists and naturalists in the ensuing centuries. However, there was no significant progress in understanding its implication and essence until Sir George Gabriel Stokes initially elaborated the relationship between light absorption and fluorescence in his seminal 1852 paper “On the Change of Refrangibility of Light”.53 He is the first person to coin the term fluorescence and propose the use of fluorescence as an analytical tool. The 20th century bore witness to ground breaking discoveries from excitation spectrum of a dye in 1905 to Stern−Volmer relation for fluorescence quenching (1919), fluorescence polarization (1923), measurement of absolute quantum yield (1924), determining fluorescence lifetime of dyes in solution (1926), quantum theory of molecular interaction (1928), Jablonski diagram (1935), quantum mechanical theory of resonance energy transfer (RET; 1948), and anisotropy (1957).48,54 These and other research provide the theoretical foundations for extensively applying fluorescence techniques in many aspects of physical, chemical, material, geological, forensic, biological, and medical sciences including fluorescent lamps, paints, tracers in hydrogeology, counterfeit detection, biosensor, intravital imaging, drug screening, DNA sequencing, clinic diagnosis, and gene therapy. It is already more than one hundred years since the earliest fluorescence-based analysis carried out by Goppelsroder (1867):55,56 the complexation of morin (a hydroxyflavone derivative) with aluminum produces a drastic enhancement of fluorescence intensity, offering a straightforward way to detect this metal. In the more than one century of developing history, fluorescent technique has been developed from initially mere content analysis to explorations in the novel, rich, and burgeoning areas such as molecular biology, cell biology, functional genomics, proteomics, medical diagnostics and treatment, and so on, which is currently the dominant analytical tool and has greatly encouraged the basic and applied studies in life sciences.57−61 At the moment, some relatively simple and convenient strategies such as FCS, FRET, time-resolved fluorescence spectroscopy, fluorescence lifetime imaging, and fluorescence in situ hybridization (FISH) are expertly used in various aspects of environics, biochemistry, and biomedicine.48,62−70 Fluorescence-based assays provide excellent spatial (diffraction limit or better) and temporal (nanosecond) resolution allowing sensitive measurement at single-molecule level to obtain structural and functional information on

3. THEORY AND BACKGROUND The optics related technology has raised both great promise and concern since its inception and introduction to the public. Thanks largely to the splendid accuracy, simplicity, diversity, and nondestructibility (clinically safe) of the method, in recent years, a large number of new applications have been developed, promoting optical technique from a primarily scientific to a more routine strategy. The most commonly applied optical methods are those based on light absorption or light emission. However, compared to absorption based methods, luminescence (generally including fluorescence and phosphorescence) is particularly important because of its high sensitivity and good specificity. The sensitivity of luminescence methods is by about 1000 times greater than that of most spectrophotometric approaches while also maintaining lower limits of detection (LODs) for desired analytes.45 Noteworthily, as a thoroughly studied and extensively applied technology in luminescence, fluorescence may be the most indispensable and fastest growing methodology of life sciences for it can provide real-time, in situ, and dynamic information on targets. Its developments allow researchers on the verge being able to deal with some of the big questions in many crucial areas such as arts, aeronautics, criminalistics, agriculture, environmentology, genetics, immunology, and oncology. Fluorescence is an optical phenomenon where absorption of a photon at appropriate energy causes the emission of another photon with longer wavelength after a brief interval called fluorescence lifetime. In general, almost all uses of fluorescence depend on the spontaneous emission of photons occurring nearly isotropically in all directions. Specifically, as illustrated in Figure 3, when the photons of visible and ultraviolet (UV) light containing energy coincident with the energy difference between two allowed energy states, molecules will absorb the light and jump from the ground state (S0) to a higher energy state (e.g., first excited singlet state S1, the second excited state S2 and so on).46−48 The molecules in excited singlet state will decay to ground state via a chain of photophysical events such as internal conversion or vibrational relaxation (loss of energy in the absence of light emission), fluorescence, intersystem crossing (from singlet state to a triplet state), and phosphorescence ensues. The discovering history of fluorescence is closely associated with the emission from plant extracts. According to Partington,49 the first recorded observation of fluorescence date back to the 16th century when N. Monardes described the peculiar blue color of the water infusion of the wood from New C

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are unavailable from organic dyes or fluorescent proteins. They are characterized by a broad excitation range, a narrow emission peak, ultrahigh brightness, greater chemical stability, and resistance to photobleaching.82,83 In comparison with organic dyes, QDs have similar quantum yields but extinction coefficients that are 10−50 times larger and significantly more reduced photobleaching rates.84 The overall effect is that QDs have 10−20 times brighter fluorescence and 100−200 times better photostability.84 Because excitons in a QD are confined in all three spatial dimensions, its electrons are quantized to certain energies, similar to that of a small molecule. Also, by binding more and more atoms together, the discrete energy levels of the atomic orbitals merge into energy bands for a semiconducting material. Therefore, semiconducting material can be regarded as a hybrid between small molecules and bulk material.85 Due to the quantum confinement effect arising from their very small (5 nm from the body, possible deterioration of their surface coating, interference of NPs retained in vital organs in the body with other imaging techniques, aggregation of NPs and deteriorated NPs in capillaries and glomarular space, interactions of NPs and their components with genetic materials, etc. need significant considerations. Although the toxicity of Au and Ag metal is smaller than that of heavy metals, their clearance from the body is still a matter of concern. Nonetheless, more studies will be needed to evaluate toxicity of

anisotropy. In principle, all liquids are isotropic: the molecular motions within the fluid are Brownian in nature and can be described by the Stokes−Einstein relationship. However, under certain circumstances a fluid may display a preferential motion or have an orientational restriction. It is then said that the fluid is structured and hence displays anisotropy.828 Anisotropy imaging is a utility anisotropy-based analysis technique that has attracted a great deal of research interest in the past few years in the field of physics, engineering, chemistry and biology. Anisotropy imaging can be performed as steady-state or timeresolved measurements in the time-domain or frequencydomain and can be used with either the scanning or wide-field method. The experimental geometry requires a polarizer or polarizing beam splitter in the detection arm for single or twodetector (simultaneous) polarization-resolved measurements, respectively. For time-resolved measurements, the excitation source must be modulated or pulsed. The detection can also be modulated or based on time-gating or time-correlated single photon counting (TCSPC).

9. CONCLUSION AND PERSPECTIVES Fluorescence is a popular measuring technique in many chemical sensors and biosensors. The properties of reporters and the range of their applications can be dramatically enhanced with the introduction of new materials of varying composition. Organic dyes can be incorporated into nanoscale particles forming more sophisticated dye-containing supermolecular structures that can be used together with lanthanide chelates, QDs, metal nanoclusters, etc.829 As for fluorescencebased technologies, it is clear that the fluorescence techniques have been able to resolve important problems concerning biological analysis in life sciences. Instruments are commercially available for measuring FRET, FISH, and FLIM, while new developments occur at an unprecedented rate, and measurements are invariably promoted by customized facilities. The application of the fluorescence techniques to three-dimensional systems, such as fully grown cells, embryos and model organisms, will be particularly crucial. Nowadays, nanoscience and nanotechnology as well as applications of nanocrystals in catalysis are being developed at a great pace all over the world. Some scientists believe nanotechnology will be the next industrial revolution, and it is ready to expand into biomedicine. Recent advances in nanoscience and nanotechnology have enabled a paradigm shift in biosensing technology. NPs are leading to the development of various biosensing devices that could be applicable to various fields of research. The integration of nanotechnology approaches into biosensors holds great promise for addressing the analytical needs of not only DNA and protein analysis but also other fields of study (e.g., heavy metal detection).830−833 The development of nanoscale materials for optical chemical sensing applications has emerged as one of the most important research areas of interest over the past decades. Nanomaterials demonstrate unique surface chemistry, distinct thermal and electrical properties, high surface area and large pore volume per mass unit area. For this reason, NPs have raised great expectations with respect to enhancing sensitivities, reducing response times, improving detection limits and using them in multiplexed systems. Optical sensors using nanomaterials open new horizons for identifying and quantifying many chemical and biochemical analytes, and they will certainly be developed as detection techniques in biomedical and environmental analysis AH

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AUTHOR INFORMATION

NPs on cells, for example, by annexin or caspase activation. In addition, more in vivo experiments should be performed to determine organ distribution and in vivo effects of NPs. The toxicity is dependent on the NPs chemical (charge on the particle) and physical (size of particle) properties, and also on the material biocompatibility. Thus, the aim of researchers is to synthesize particles that are biocompatible and are cytotoxic only at very high concentrations. In addition, ligands are essential to the reduction of cytotoxicity of many NPs, as uncoated NPs are generally cytotoxic themselves.834−836 It is likely that significant quantities of NPs will be taken up by phagocytes and macrophages, so they must be protected from the harsh chemical environments present through the employment of a chemically inert shell.837 Another problem is the delivery of the nanobioconjugate inside the live cells in the intact form. The application potential of several new nanomaterials should be explored and current coating chemistry should be extended for them. These materials include highly fluorescent doped semiconductor NPs,838−841 fluorescent gold and silver clusters,842 fluorescent carbon NPs843 or other carbon materials,844,845 and fluorescent silicon nanocrystals,846 These materials are expected to be less toxic nanoprobes. When toxicity is observed, it has been found to arise from parameters that are not currently controllable, such as NP stability or dose. Therefore, it is unlikely that toxicity issues should hinder the development of the exciting bioapplications presented in this field. Nanomaterials possess strong potentials with reference to biosensing and bioimaging, providing improvements in detection sensitivity, assay simplicity, and low-cost methods. Their performances can be enhanced by improving their purity and narrowing their size/shape/conjugation distributions, which can be achieved with various separation techniques. Further progress is expected to push detection limits further into the subfemtomolar range (or lower) with noninvasive and nondegenerative NPs and lead to development of new assay formats for multiplex determinations. NPs have been exploited in several areas of biosensing and bioimaging, including immunohistochemistry, microarray technologies and advanced fluorescence techniques such as FISH, and in vivo fluorescence imaging using practical techniques and multiphoton microscopy. Although NP-based techniques are relatively new, they are reinforced year by year and are showing to be novel alternatives to several other biosensing systems, opening new horizons for biological research and applications. It is anticipated that progresses in nanosciences, combined with the alluring features of many NP systems, will render these particles cumulatively fascinating for bioanalytical applications in the future. In conclusion, nanoscience is an interdisciplinary region covering biology, chemistry, physics, materials, and engineering. Consequently, the field of nanosensors will carry on involving mutual reciprocities and cooperations among chemists, physicists, engineers and material scientists. Future discoveries and applications of nanoscale sensing devices will proceed to evolve, in turn, greatly improving the quality of daily life and likely benefiting various fields including: industrial sectors (chemical and electronic industries), manufacture, medicine and health, environmental monitoring, military and national defense as well as network and communications. Thus, nanomaterials could provide a great contribution to the development of analytical science, and also benefit to a large extent from modern analytical chemistry.

Corresponding Author

*Phone: +86-28-85418180. Fax: +86-28-85412316. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Jun Yao studied Chemistry at China West Normal University, where he obtained his B.S. degree in Chemistry in 2006. Subsequently he worked in the field of bioanalytical chemistry, receiving his M.S. degree from the same university in 2009. Following graduate studies, he became a university teacher of chemistry and pharmacy at Chongqing Three Gorges Medical College, China. Between 2010 and 2013, he joined the Research Center of Analytical Instrumentation, Analytical and Testing Center of Sichuan University as a Ph.D. candidate. After 2013, he is a lecturer in Southwest Petroleum University, China and researches biochemical and biomedical analysis based on nanomaterials and spectroscopy related technology, especially molecular fluorescence technique. His research interests involve inorganic nanomaterials and multifunctional nanostructures, spanning from synthetic methodology to device fabrication, mainly focusing on a variety of biochemical and biomedical applications of fluorescent nanomaterials such as biosensor, bioimaging, drug delivery, medical diagnostics and biotherapy. His other research interests are in the area of chromatography, mass spectrometry and hyphenated techniques.

Mei Yang was born in Sichuan, China, and studied chemistry at China West Normal University. As an undergraduate, she conducted research on the development of new biosensors and their applications in biomolecule analysis such as amino acid and protein. After receiving her B.S. degree in chemistry in 2010, she joined the Research Center of Analytical Instrumentation, Analytical and Testing Center of AI

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DOX doxorubicin ds-DNA double-stranded DNA EDC 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride EGF epidermal growth factor EGFP enhanced green fluorescent protein ELISA enzyme linked immunosorbent assay FCS fluorescence correlation spectroscopy FDA Food and Drug Administration FISH fluorescence in situ hybridization FLIM fluorescence lifetime imaging microscopy FP fluorescence polarization FPIA fluorescence polarization immunoassay FRET fluorescence resonance energy transfer GAH−IgG goat antihuman immunoglobulin G GE gel electrophoresis GO graphene oxide HBsAg hepatitis B surface antigens HER2 human epidermal growth factor receptor 2 HGP Human Genomic Project H−IgG human IgG H−IgG−NaYF4: Yb, Er human IgG-modified NaYF4: Yb, Er HNSCC head and neck squamous cell carcinoma HAS human serum albumin HTS high throughput screening IgG immunoglobulin G ITC isothermal titration calorimetry LODs limits of detection LSPR localized surface plasmon resonance MBP maltose binding protein MEF metal-enhanced fluorescence MNPs magnetic nanoparticles MRI magnetic resonance imaging NIR near-infrared NIRF near-infrared fluorescence NMR nuclear magnetic resonance NPs nanoparticles ORMOSIL organically modified silica PC protein corona PCR polymerase chain reaction PDT photodynamic therapy PEG poly(ethylene glycol) PET positron emission tomography PL photoluminescence PS photosensitizer PSMA prostate-specific membrane antigen PTT photothermal therapy QDs quantum dots QCM quartz crystal microbalance RAG−IgG rabbit antigoat IgG RAG−IgG−AuNPs rabbit antigoat IgG-modified AuNPs RE rare earth RET resonance energy transfer RISCs RNA-induced silencing complexes RNAi RNA interference ROS reactive oxygen species SELEX systematic evolution of ligands by exponential enrichment SERS surface-enhanced Raman spectroscopy SIFs silver island films siRNA small interfering RNA SNP single nucleotide polymorphism SPECT single photon emission computed tomography

Sichuan University. Her research interests are in the area of bioanalytical chemistry. Current work focuses on green and morphology controlled nanomaterials synthesis, the functionalization of inorganic nanoparticles with inorganic and/or organic ligands, and integration of these novel materials with molecular fluorescent technique. Her other scientific interests include the applications of HPLC and LC-MS on medical diagnostics.

Yixiang Duan received his B.S. degree from Fudan University and M.S. degree in analytical chemistry from Changchun Institute of Applied Chemistry, Chinese Academy of Science in 1988 and Ph.D. degree in analytical chemistry jointly from Jilin University, China, and Indiana University, USA in 1994. Then he did his postdoctoral research at Los Alamos National Laboratory. From 1997 to 2010, he was a Principal Investigator and Staff Scientist with the Chemical Diagnostics and Engineering Group, Los Alamos National Laboratory. He is currently a National Special Professor and Director of Research Centre of Analytical Instrumentation, Sichuan University, China. His current research interests include nanomaterial-based optical biosensors, molecular spectrometry, noninvasive medical diagnostics, novel analytical instrumentation, as well as various applications of surface chemistry to nanomaterials, nanoscience, and biological science.

ACKNOWLEDGMENTS The authors are grateful to the financial support from National Major Scientific Instruments and Equipment Development Special Funds (No.2011YQ030113), National Recruitment Program of Global Experts (NRPGE), the Hundred Talents Program of Sichuan Province (HTPSP), and the Startup Funding of Sichuan University for setting up the Research Center of Analytical Instrumentation. ABBREVIATIONS AFP α-fetoprotein AβF amyloid beta fibrillation Ag NCs silver nanoclusters Alexa-IgG Alexa Fluor-labeled antirabbit IgG ATP adenosine triphosphate AuNPs gold nanoparticles CD circular dichroism CNTs carbon nanotubes CaDPA calcium dipicolinate C-dots carbon dots Con A concanavalin A CT computed tomography DCS differential centrifugal sedimentation DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid AJ

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SPIONs superparamagnetic iron oxide nanoparticles ss-DNA single-stranded DNA SWCNTs single-walled carbon nanotubes TCSPC time-correlated single photon counting TEM transmission electron microscopy TNB trinitrobenzene TNT 2,4,6-trinitrotoluene TRITC tetramethylrhodamine isothiocyanate UCNPs upconversion nanoparticles UCS-Ball upconversional sesame ball UV ultraviolet UV−vis ultraviolet−visible VEGF vascular endothelial growth factor β-CD β-cyclodextrin

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