Toward Biocompatible Semiconductor Quantum Dots: From

Review pubs.acs.org/CR

Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application Juan Zhou,‡,§ Yong Yang,§ and Chun-yang Zhang*,†,§ †

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China ‡ State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China § Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China S Supporting Information *

3.1.2. Multicolor, Multimodal, and Multiphoton Imaging 3.1.3. Near-Infrared (NIR) Quantum Dots for Deep Tissue Imaging 3.2. Quantum Dot-Based Biobarcodes 3.3. Quantum Dot-Based Biosensors 3.3.1. Quantum Dot-Based Conventional Biosensors 3.3.2. Quantum Dot-Based Energy Transfer Sensors 3.3.3. Single Quantum Dot-Based Nanosensors 3.4. Drug Delivery and Cancer Therapy 3.4.1. Single Quantum Dot-Based Drug Delivery System 3.4.2. Mesoporous Silica/Quantum Dot-Based Drug Delivery System 3.4.3. Liposome/Quantum Dot-Based Drug Delivery System 3.4.4. Polymeric Micelle/Quantum Dot-Based Drug Delivery System 4. Conclusions and Perspectives Associated Content Supporting Information Author Information Corresponding Author Notes Biographies Acknowledgments References

CONTENTS 1. Introduction 2. Preparation of Biocompatible Quantum Dots 2.1. Synthesis of Quantum Dots in Biological Systems 2.1.1. Synthesis of Quantum Dots in Living Yeast Cells 2.1.2. Synthesis of Quantum Dots in Escherichia coli 2.1.3. Synthesis of Quantum Dots in Other Living Organisms 2.2. Synthesis of Quantum Dots in Biomimetic Systems 2.2.1. Synthesis of Quantum Dots in Artificial Biomimetic Systems 2.2.2. Synthesis of Quantum Dots Using Biomolecules as Templates 2.3. Functionalization of Chemically Synthesized Quantum Dots with Biomolecules 2.3.1. Water Solubilization of Qauntum Dots 2.3.2. Covalent Conjugation Strategy 2.3.3. Electrostatic Interactions 2.3.4. Ligand−Receptor Interaction 2.3.5. Metal-Affinity Coordination 2.3.6. Click Reaction 2.3.7. Virus Encapsulation 3. Biomedical Applications of Semiconductor Quantum Dots 3.1. Biomedical Labeling and Imaging 3.1.1. Quantum Dots for Targeted Biolabeling and Bioimaging © XXXX American Chemical Society

A B B D E F F H H J J L N N N O O

Q R S U V W AA AC AC AD AE AE AG AG AG AG AG AG AG AH AH

1. INTRODUCTION The understanding of biological systems at the molecular scale is increasingly reliant on the capability of tracking miscellaneous biomolecules and their events with improved spatial and temporal resolution.1 The discovery of green fluorescent protein (GFP) and its various mutants2 and the emergence of numerous organic dyes3,4 have provided essential tools that

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Received: April 26, 2015

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DOI: 10.1021/acs.chemrev.5b00049 Chem. Rev. XXXX, XXX, XXX−XXX

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allow for the investigation of complex processes in cells. The development of photoswitchable/photoactivatable fluorescent proteins5−7 and organic fluorophores,8−10 in concert with the high-accuracy localization of individual fluorescence spots, enables high-resolution imaging of intracellular molecules with near molecular-scale resolution (20 nm or less).11,12 However, the application of fluorescent proteins and dyes are often limited in cases where small amounts of biomolecules are present and long-term imaging is required.13 The development of novel nanomaterials, especially semiconductor quantum dots (QDs), is considered a valuable supplement to the conventional fluorescent proteins and organic dyes. The QDs usually refer to the II−VI, III−V, and IV−VI binary and their alloyed semiconductor materials with a confined size on the nanoscale (2−20 nm) in three dimensions. Because of a number of distinguished characteristics, including (i) prominent optical performance with good photo-stability,13−15 high quantum yield (QY) and long fluorescence lifetime,13 (ii) simultaneous excitation of multiple QDs by a single light source,16 (iii) narrow, symmetrical, and size-tunable emission spectra coupled with wide absorption spectra,17 and (iv) broad spectral windows spanning from the ultraviolet to infrared region,13 the QDs have attracted tremendous attention from materials scientists, physicists, chemists, and biologists worldwide. Benefiting from the interdisciplinary research among nanotechnology, chemical processing, biotechnology, and systems engineering, tremendous progress has been made in the biomedical applications of QDs over the past two decades since the first studies regarding their biological applications were reported in 1998.18,19 The QDs can be prepared by means of physical, chemical, and biological approaches, depending on the synthesis platform involved. The physical synthesis methods, such as laser physical vapor deposition20 and laser irradiation of large particles,21−23 are generally facile, time-saving, and eco-friendly. However, the resulting QDs usually harbor high surface defects, poor stability, and low quantum yield.24,25 The chemical synthesis routes, including the organic-phase and water-phase synthesis methods,26−30 are the classical approaches to prepare QDs with monodispersity and good optical performance. Even though significant progress has been made in simplifying the synthesis routes,31−33 improving quantum yields,34,35 controlling the size and shape, and elucidating the mechanisms of QD formation,36 the chemical synthesis strategies usually rely on harsh reaction conditions37 and toxic organic solvents to produce fluorescent products.38 Moreover, surface modification is required for the achievement of desirable functions. Alternatively, the biosynthesis methods can not only carry out the synthesis reaction in mild conditions, but also endow the QDs with inherent biostability and biocompatibility without resorting to additional cap exchange and encapsulation treatment,38,39 thereby offering a green synthesis route to prepare biocompatible QDs. Similar to the chemically synthesized QDs, the biosynthesized QDs display size-tunable emission, simultaneous excitation of different-sized QDs by a single light source, and component-tunable broad spectral windows (Figure 1). In this review, we focused on (i) recent progress in the synthesis of biocompatible QDs by using living organisms and biomimetic systems, (ii) effective approaches to functionalize the chemically synthesized QDs with various biomolecules, and (iii) applications of biocompatible QDs in biomedical fields ranging from in vitro biosensing to in vivo

Figure 1. Photophysical properties of the QDs synthesized in biological/biomimetic systems. (A) Size-tunable fluorescence emission and simultaneous excitation of multiple QDs by a single light source. (B) The QDs with different components show broad spectral windows spanning from the ultraviolet to infrared region. It should be noted that these material combinations are only examples and that many others exist.

bioimaging and drug delivery. Finally, we discuss several issues that need to be further investigated in the future.

2. PREPARATION OF BIOCOMPATIBLE QUANTUM DOTS Biocompatibility is an essential prerequisite for the use of QDs in biological and biomedical research. The preparation of QDs in biological and biomimetic systems may endow the pristine QDs with good biocompatibility. However, for most chemically synthesized QDs, especially organic-phase QDs, the major challenge is their nonbiocompatibility, which restricts their direct applications in biological and biomedical research. For addressing this issue, great efforts have been made toward water-solubilization and biofunctionalization of chemically synthesized QDs.40−42 This review focuses on the preparation of biocompatible QDs, including (i) the synthesis of QDs in biological systems, (ii) the synthesis of QDs in biomimetic systems, and (iii) the functionalization of chemically synthesized QDs with biomolecules. 2.1. Synthesis of Quantum Dots in Biological Systems

To date, a number of nanomaterials have been successfully synthesized within various biological organisms.43−47 The employment of biological systems as the reaction platforms has three distinctive features: (i) intrinsic generation of metalreducing and -binding agents (e.g., amino acids, proteins, and chelating peptides) with the capability of reducing metal ions, decomposing metallic compounds, accumulating metals, and protecting the products, (ii) easy modulation of the platform by means of genetic engineering for the expression of specific biomolecules to regulate the QD growth, size distribution, and fluorescence emission, and (iii) moderate reaction conditions without the involvement of high temperature, rigorous deoxygenation, or toxic organic solvents. The revelation of natural metal-related biodetoxification, biomineralization, and enzymatic reactions really facilitates the B


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Table 1. Biosynthesis of QDs in Various Organisms materials

organisms

intracellular/ extracellular

size distribution (nm)

absorption/emission wavelength (nm/nm)

quantum yield

ref

318/384 (excitation at 320) (engineered JM109 strain) 254/330 (excitation at 260) (engineered R189 strain) 300 and 315/not reported (intracellular CdS) 365/not reported (extracellular CdS)

0.007%

39

not reported

56

CdS

engineered E. coli

intracellular

CdS

Candida glabrata, Schizosaccharomyces pombe engineered E. coli engineered E. coli tobacco mosaic virus bacterial cellulose nanofiber Rhodopseudomonas palustris Brevibacterium casei SRKP2 Phanerochaete chrysosporium Fusarium oxysporum Torulopsis sp. Saccharomyces cerevisiae MTCC 2918 E. coli curli fibrils sulfate-reducing bacteria Saccharomyces cerevisiae Gsh A-overexprssed E. coli E. coli Fusarium oxysporum earthworm living yeast cells engineered yeast cells yeast cells Staphylococcus aureus Veillonella atypica Helminthosporum solani Shewanella oneidensis HepG2 cells

intracellular extracellular

2−6 (engineered JM109 strain) 3−4 (engineered R189 strain) 2 ± 0.3 2.9 ± 0.5

intracellular intracellular in vitro in vitro

2−5 6 (average size) 5 8

not reported/not reported 410/445−510 not reported/not reported 426/417 and 437

not not not not

reported reported reported reported

62 67 69 71

intracellular

8.01 ± 0.25

425/not reported

not reported

72

in vitro

10−30

370/430 (excitation at 370)

not reported

76

extracellular

1.5−2.0

296−298/458

not reported

81

extracellular intracellular intracellular

5−20 2−5 30−40

not reported not reported not reported

83 57 58

extracellular in vitro extracellular intracellular

∼5 2−5 2.0−3.6 2−3

∼450/not reported ∼330/not reported 302.57/350 and 440 (excitation at 280) 302.57/ 380 and 440 and 525 (excitation at 325) not reported/490 (excitation at 405) not reported/not reported 440−510/490−560 300−500/450 (excitation at 350)

not reported not reported ∼33% not reported

66 77 60 65

extracellular extracellular in vivo intracellular intracellular intracellular intracellular extracellular extracellular extracellular in vitro

2.0−3.2 15−20 2.33 ± 0.59 2.69 ± 0.07 not reported not reported 1.8 ± 0.5 2.3 ± 1.3 5.5 ± 2 9 ± 3.5 5.45 (average size) 3−5

∼430−480/488−551 400−450/475 (excitation at 400) 450/520 not reported/520 to 560 to 670 ∼520/575 not reported/not reported not reported/520 314 and 334/510−531 (excitation at 365) 350/430 (excitation at 380) 410/not reported 744/945

15% not reported 8.3% not reported 4.7% not reported not reported not reported 1% not reported 1.56 ± 0.21%

68 82 85 38 59 61 79 74 80 75 84

not reported/∼425 (CdS, excitation at 350)

not reported

70

10−25 (CdS) 65 (ZnS) 2−4 (CdSe) 3−6 (ZnSe) 10.01 ± 0.97 (CdZn)

420/not reported 400/not reported not reported/512 not reported/368 420/not reported

not reported

73

10.33% (CdSe)

78

not reported

64

5.01 ± 0.89 (CdSe) 7.01 ± 0.66 (CdTe) 3.92 ± 0.35 (SeZn) 10.35 ± 0.68 (CdZn)

380/560−615 (CdSe, excitation at 488)

CdS CdS CdS CdS CdS CdS CdS CdS PbS ZnS ZnS ZnS CdTe CdTe CdTe CdTe CdTe CdSe CdSe CdSe CdSe CdSe CdSe Ag2S Ag2S ZnS, CdS ZnS,CdS

engineered M13 bacteriophage Klebsiella pneumoniae

in vitro extracellular

ZnSe, CdSe

Veillonella atypica

extracellular

CdZn, CdSe, CdTe, SeZn

engineered E. coli

intracellular E. coli DH5a (pTJ1AtPCS)

intracellular E. coli DH5a (pTJ1PpMT)

(CdS) (ZnS) (CdSe) (ZnSe) (CdZn)

4.99 ± 0.69 (CdSe) 5.83 ± 1.60 (CdTe) 3.95 ± 1.12 (SeZn)

understanding of nanomaterial formation mechanisms in biological systems. First, the biodetoxification is an intrinsic

behavior of many organisms in response to heavy metal ions (e.g., cadmium ion), enabling the conversion of heavy metal C


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ions to either low-valence metal compounds or metal-protein complexes (e.g., phytochelatin and metallothionein).47,48 Second, the biomineralization process often undergoes a series of physiological actions in many microorganisms, offering an efficient approach to immobilize metal ions onto solid-phase inorganic materials.49 Third, the enzymatic reaction plays important roles in the growth of QDs.50 For example, acetyl choline esterase hydrolyzes acetylthiocholine into thiocholine, which accelerates the decomposition of sodium thiosulfate to yield hydrogen sulfide, and the subsequent reaction of hydrogen sulfides with cadmium ions may lead to the generation of CdS QDs.51 A number of enzymatic reactions with the capability of producing hydrogen sulfide, such as the hydrolyzation of thiophosphate by alkaline phosphatase, the catalytic oxidation of 1-thio-β-D-glucose by glucose oxidase, and the decomposition of S-adenosyl-L-homocysteine by Sadenosyl-L-monocysteine hydrolase and methionine γ-lyase, may be used to generate CdS QDs.51−53 Additionally, the enzymatic reactions can produce some small biomolecules (e.g., glutathione and phosphate), which may function as surface ligands to promote the stabilization of QDs.54,55 These discoveries offer important clues for producing and harvesting QDs in living organisms. In view of the practical applications of QDs in biological and medical research, immediate biosynthesis strategies with the capability of producing biocompatible QDs are highly preferred. Ideally, the obtained semiconductor nanocrystals should be water-soluble, stable in a physiological environment, fluorescent in biological milieu, easily taken up by cells, nontoxic to the body, and able to specifically label target molecules. The use of living organisms as the synthesis machines can meet these stringent criteria to some extent (especially the solubility and stability of the obtained QDs). In addition, the biosynthesis strategy can avoid the extreme conditions that are often implicated in chemical synthesis reactions (e.g., anaerobic reaction conditions, high temperature, and toxic substrates), offering a green route for the preparation of biocompatible QDs. To date, a variety of QDs have been successfully prepared in different organisms ranging from prokaryotes to eukaryotes and even living animals, and the primary properties of the obtained QDs are summarized in Table 1. 2.1.1. Synthesis of Quantum Dots in Living Yeast Cells. Dameron and Winge demonstrated for the first time the synthesis of CdS semiconductor crystallites in a biological system in 1989.56 They obtained intracellular CdS crystallites by incubating the yeasts Candida glabrata and Schizosaccharomyces pombe with cadmium salts. The cadmium ions stimulated the yeast cells to produce metal-chelating peptides with a primary structure of γ-(Glu-Cys)n-Gly, which in turn combined with cadmium ions and reacted with the intracellular sulfide to yield the CdS crystallites. This approach is very simple and easily manipulated, but it is only suitable for the production of metal sulfide crystallites. Subsequently, PbS57 and ZnS58 QDs were prepared by feeding yeast Torulopsis and Saccharomyces cerevisiae with lead salts and zinc sulfate salts, respectively. Notably, the combination of the detoxification reaction with the intracellular metabolism pathway may provide an efficient way to prepare different kinds of QDs in living organisms. By coupling two intracellular biochemical reactions at appropriate time and space, Pang and Xie produced fluorescent CdSe QDs in yeast cells of Saccharomyces cerevisiae (Figure 2).38 When incubated with stationary-phase yeast cells,

Figure 2. (A) Biosynthesis process of fluorescent CdSe QDs in living yeast cells. (B) Fluorescent image of biosynthesized QDs in yeast cells. Figures adapted from ref 38. Copyright 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Na2SeO3 undergoes an intrinsic glutathione metabolic pathway to yield the organoselenium precursor, whereas CdCl2 undergoes a detoxification pathway to yield the organocadmium precursor. The subsequent reaction of Se precursors with Cd precursors in cytoplasm and mitochondria may generate CdSe QDs, and the emission wavelength of the obtained QDs can be facilely tuned by adjusting the incubation time of seleniumized yeast cells with CdCl2. For the biosynthesis of fluorescent QDs, it is very important to improve both their yield and optical properties. Because of their diversity and easy accessibility, the yeast cells may function as effective bioreactors to synthesize a variety of QDs. Pang and Xie successfully prepared CdSe QDs with relatively high yield and QY (4.7%) using genetically engineered yeast cells.59 Overexpression of γ-glutamylcysteine ligase in genetically engineered yeast cells facilitates the production of large amounts of glutathiones.59 Besides the intracellular metabolic process, the QDs may also be prepared through an extracellular growth mechanism. Zhao and colleagues produced proteincapped CdTe QDs by culturing the yeast cells with Cd and Te precursors (Figure 3).60 The obtained CdTe QDs have tunable

Figure 3. Extracellular growth pathway (A) and endocytosis pathway (B) of protein-capped CdTe QDs. The endocytosis pathway involves the adherence of QDs to the cell membrane (I), endocytosis of QDs into the cell (II), release of QDs from the endosome into the cytoplasm (III), and entrance of QDs into the nucleus (IV). Figure reprinted from ref 60. Copyright 2010 Springerlink.

fluorescence emission, uniform size distribution (2−3.6 nm), excellent yield (∼90%), and high QY (33%). The high quantum yield may be attributed to the addition of a reducing reagent (sodium borohydride) and thiol reagent (mercaptosuccinic acid) to the medium. Notably, the biochemical reaction may significantly influence the mechanical strength D


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and the structure of yeast cells.61 The introduction of Cd2+ can decrease the nanomechanical strength of yeast cells because Cd2+ may induce reactive oxygen species and become toxic to the cells. In contrast, the coexistence of Na2SeO3 can significantly improve the nanomechanical strength of yeast cells because the generation of CdSe QDs in the cells may detoxify Cd2+.61 2.1.2. Synthesis of Quantum Dots in Escherichia coli. In addition to eukaryotic cells, some prokaryotes may also be employed to generate fluorescent nanomaterials. Escherichia coli (E. coli) features a single-compartment structure, which may precisely tailor the size and crystallinity of QDs. In addition, the relatively simple cellular metabolism in E. coli is beneficial for the elucidation of the QD formation mechanism in the cells. Iverson et al. demonstrated the use of E. coli for the synthesis of QDs in 2004.62 They administrated a mixture of cadmium and sulfide precursors into the stationary-phase E. coli cells to produce the CdS nanocrystals.62 The obtained nanocrystals are polydisperse with a size distribution of 2−5 nm. Notably, some parameters, such as growth phase, strain type, genetic and physiologic factors, and capping agents may strongly affect the nanocrystal nucleation, and can be used to modulate the particle size, shape, and crystal structure. This work opens a new avenue for the use of bacteria to synthesize unnatural materials.63 Inspired by the biodetoxification mechanism in yeast cells, Chen and colleagues genetically recombined E. coli cells to express phytochelatin synthase for the synthesis of CdS nanocrystals.39 The engineered bacterium may act as an ecofriendly biofactory to produce water-soluble CdS nanocrystals whose size distribution may be simply tuned by different strains. The nanocrystals obtained in the engineered JM109 strain show a polydispersed size distribution of 2−6 nm due to the use of a heterogeneous population of phytochelatins (i.e., PC2, PC3, and PC4) as the capping agents. In contrast, the nanocrystals obtained in the engineered 189 strain display a uniform size distribution of 3−4 nm due to the use of a homogeneous PC4 population as the primary capping agent. Nevertheless, the QY of the obtained CdS nanocrystals is very low (0.007%). Similarly, Lee et al. produced a number of metal nanoparticles by using the recombinant E. coli cells as the biosynthesis machines.64 The recombinant E. coli cells may express either phytochelatin synthase (PCS) and metallothionein (MT) individually or both simultaneously. By incubating the engineered E. coli with the corresponding metal ions, four kinds of QDs (i.e., CdZn, CdSe, CdTe, and SeZn QDs) with different sizes can be synthesized in the cells (Figure 4). In particular, the particle size can be controlled by simply adjusting the concentration of metal ions. When the concentration of Cd2+ and Se+ is adjusted from 0.5 to 5.0 mM, the diameter of CdSe QDs may be tuned from 3.31 ± 0.43 nm to 5.10 ± 0.57 nm. Notably, the coexpression of PCS and MT in the cells may produce the CdSe QDs with 4-fold fluorescence enhancement as compared with those obtained in the cells expressing PCS/MT alone. Several new types of fluorescent metal nanoparticles (e.g., SrGd, SrPr, and PrGd) have been produced by incubating the cells with the corresponding metal ions.64 The obtained QDs can be used for the imaging of human fibroblast cells. Recently, PérezDonoso and colleagues synthesized CdTe QDs by overexpressing the glutathione biosynthesis-associated enzymes in E. coli and incubating the recombinant cells with K2TeO3 and CdCl2.65 With the development of genetic engineering techniques, more and more specific proteins and peptides can

Figure 4. TEM images of CdZn, CdSe, CdTe, and SeZn QDs synthesized in vivo by recombinant E. coli cells expressing phytochelatin synthase (PCS) and metallothionein (MT). CdZn (A), CdSe (B), CdTe (C), and SeZn (D) QDs synthesized in recombinant E. coli cells expressing Arabidopsis thaliana PCS (AtPCS). The nanocrystals with interplanar distances of 4.4, 3.51, 3.00, and 2.82 Å correspond to {111} face-centered-monoclinic, {111} cubic, {102} hexagonal, and {012} monoclinic structures, respectively. CdZn (E), CdSe (F), CdTe (G), and SeZn (H) QDs synthesized in recombinant E. coli cells expressing Pseudomonas putida MT (PpMT). The nanocrystals with interplanar distances of 2.96, 2.85, 3.69, and 2.25 Å correspond to {221} monoclinic, {121} orthorhombic, {111} cubic, and {111} cubic structures, respectively. CdZn (I), CdSe (J), CdTe (K), and SeZn (L) QDs synthesized in recombinant E. coli cells expressing both AtPCS and PpMT. The nanocrystals with interplanar distances of 2.82, 3.51, 3.22, and 3.25 Å correspond to {221} monoclinic, {111} cubic, {111} cubic, and {111} cubic-plane structures, respectively. Figures reprinted from ref 64. Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

be expressed in E. coil cells for the regulation of QD growth.66,67 The growth of QDs in living organisms relies on intrinsic cellular metabolism products rather than exogenous chemical reduction/coupling reagents, thereby endowing the QDs with both inherent biocompability and reduced toxicity. Although the intracellularly prepared QDs have the capability of in situ bioimaging, they usually require subsequent isolation from the organisms prior to their biomedical applications. In this regard, the growth of QDs from either the secreted metabolites of E


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organisms or extracellular biomolecules is an alternative method that is worth exploring. Importantly, the properties of QDs can be facilely regulated by altering the extracellular environment. Li and colleagues demonstrated a one-step extracellular synthesis approach to prepare CdTe QDs in Luria−Bertani medium.68 By adjusting the incubation time of Cd and Te precursors with E. coli cells/E. coli-secreting proteins, a series of QDs with tunable fluorescence emission ranging from 488 to 551 nm can be obtained. The QY may reach up to 15%. In particular, the proteins secreted by E. coli play important roles in both the growth of QDs and the reduction of toxicity. It should be noted that the extracellular synthesis approach often involves additional chemical reduction/coupling reagents (e.g., sodium borohydride and mercaptosuccinic acid) to promote the QD growth. 2.1.3. Synthesis of Quantum Dots in Other Living Organisms. Because of the diversity of species in the world, a large number of biological organisms can be domesticated to prepare semiconductor QDs with the involvement of similar biosynthesis mechanisms in yeasts and E. coli strains. For example, the tobacco mosaic virus (TMV) contains abundant protein subunits with multiple charged amino acid residues and has the capability of nucleating CdS and PbS nanocrystals.69 The engineered M13 bacteriophage can express semiconductor-nucleating peptides on the viral capsid and may assemble ZnS and CdS nanowires with the preferred orientation.70 The bacterial cellulose has a large surface area with many hydroxyl and ether groups, which provides abundant active sites to adsorb metal ions for QD nucleation.71 The photosynthetic bacteria Rhodopseudomonas palustris may offer a S source via intracellular cysteine desulfhydrase to produce CdS QDs in the presence of cadmium.72 Notably, some bacteria exhibit a strong reducing capability toward the semiconductor precursors.73−77 For example, the sulfate-reducing bacteria Desulfobacteriaceae is able to aggregate the metal ions (e.g., Zn ions) to generate ZnS deposits in a biofilm.77 Veillonella atypica can reduce Se (IV) to Se (II), which may react with zinc/cadmium ions to yield ZnSe/CdSe QDs.74,78 The metal-reducing bacterium Shewanella oneidensis can reduce AgNO3 to produce Ag2S nanocrystals in the presence of S2O3− ions. The proteins/peptides secreted by the bacterium may function as surface stabilizers, and the resulting Ag2S QDs display no toxicity toward either prokaryotic cells (e.g., E. coli cell, Shewanella oneidensis, and Bacillus subtilis) or eukaryotic cells (e.g., mouse lung epithelial and macrophage cells).75 Recently, Pang et al. demonstrated a space-time coupling strategy to prepare fluorescent CdSe QDs in Staphylococcus aureus cells.79 This strategy involves (i) the reduction of Na2SeO3 through the intrinsic glutathione metabolic pathway to form a Se precursor, (ii) the intracellular detoxification of Cd2+ to form a Cd precursor, and (iii) the use of endogenous mercapto-containing biomolecules to stabilize the QDs.79 In addition, some funguses (e.g., Fusarium oxysporum, Phanerochaete chrysosporium, and helminthosporum solani),80−83 cancer cells,84 earthworms,85 and even the liver and kidneys of rats86,87 can be used to produce luminescent QDs. Wan and colleagues prepared near-infrared (NIR) Ag2S QDs by feeding hepatoma carcinoma (HepG2) cells with silver nitrate and sodium sulfide (Figure 5).84 The high concentration of the reduced glutathione in the cells allows for the formation of stable Ag2S nanocrystals. The obtained Ag2S nanocrystals are eligible for both in vitro and in vivo imaging applications.84 Green and colleagues extended the biosynthesis systems from

Figure 5. Intracellular synthesis of near-infrared (NIR) Ag2S QDs by culturing hepatoma carcinoma (HepG2) cells with silver nitrate and sodium sulfide. Bright-field image of HepG2 cells (A), NIR images of HepG2 cells immediately (B) and 16 h (C) after uptake of silver nitrate and sodium sulfide. (D) Structural diagram of Ag2S QDs stabilized by the reduced glutathione. Figures adapted from ref 84. Copyright 2013 American Chemical Society.

simple cells to the superior organism of the earthworm. They took advantage of the endogenous heavy-metal detoxification process in earthworms and obtained the luminescent and water-soluble CdTe QDs by exposing the earthworms to CdCl2 and NaTeO3 salts (Figure 6).85 Interestingly, the synthesis of

Figure 6. Anatomy of the worm and regions for both the generation of metallothionein and synthesis of QDs (A−C) as well as optical characterization of the QDs (D, E). (A) The earthworm Lumbricus rubellus. The posterior alimentary canal (PAC) is defined as the gut section following the thickened glandular clitellum. (B) Cross-section of the posterior section of the earthworm with the chloragogenous tissue surrounding the PAC (marked by arrows). (C) Metallothionein predominantly locates in the chloragogenous tissue, as determined by immunoperoxidase histochemistry with an earthworm-specific metallothionein antibody (marked by arrows). (D) Absorption (dotted line) and emission (solid line) spectra of CdTe QDs obtained from choragogen cells. Inset: (top) emission spectra of original (blue) and aged (7 days, black) CdTe QDs and (bottom) photograph of original CdTe QDs at 365 nm excitation. (E) Emission lifetime measurement of freshly isolated CdTe QDs. Figure adapted from ref 85. Copyright 2013 Macmillan Publishers Limited.

QDs inside the body does not affect the growth of the earthworms, and the resulting CdTe QDs can be used directly for bioimaging without further surface modification.85,88 Moreover, the CdS and CdSe QDs can be obtained by administering rat liver and kidneys with subacute cadmium.86,87 2.2. Synthesis of Quantum Dots in Biomimetic Systems

In living organisms, some specific biomolecules (e.g., metalbinding peptides and proteins) play critical roles in the F


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Table 2. Synthesis of QDs in Biomimetic Systems materials

biomimetic templates

size distribution (nm)

CdS CdS CdS

lipid vesicles DNA plasmid tRNA

CdS CdS CdS CdS CdS

phosphorothioate oligoG10 bacterial S layers apoferritin achymotrypsin pepsin

5−6 4.0 ± 0.6 4.4 ± 0.4 5.5 ± 1.0 5 5 1.5−7.2 3.0 ± 0.7 1.7−5.2

CdS CdS PbS PbS PbS PbS Ag2S Ag2S Ag2S HgS

pepsin peptide-amphiphile nanofibers DNA nucleotide thrombin-binding aptamer luciferase nanopore cucurbit[7]uril bovine serum albumin ribonuclease-A bovine serum albumin

6.3 3−5 4 ∼4 3−6 ∼4 20 1.6−6.8 5.6 20−40

Ag2Se

(1.5 ± 0.4)− (2.4 ± 0.5)

CdTe CdTe CdTe

glutathione, glutathione reductase, reduced nicotinamide adenine dinucleotide phosphate gelatin polymer DNA DNA

CdTe CdTe CdTe CdTe ZnTe

DNA ribonuclease-A glutathione dithiol-peptide, trypsin poly(amidoamine) dendrimers

not reported 4.8 ∼6 (average size) not reported ∼2.7 3−6 2.5−5.5 2.9−6.0

CdSe CdSe CdS, CdSe

hydrogel polymer gelatin polymer bovine serum albumin

5.00 ± 0.79 tunable size 3.8−7.0

CdTe, CdZnTe

RGD peptides

2.73−3.56 (CdTe) 2.96−3.50 (CdZnTe) 4−5 (CdSe) ∼5 (ZnS) 12 (CdSe/ ZnS) 3.5 ± 0.3 3.8 ± 0.4 8.5 ± 1.3 (Ag2Se)

CdSe/ZnS

bifunctional peptide

CdTe: Zn2+ Mn: ZnS MxSey (M = Ag, Cd, Pb, Cu)

DNA bovine serum albumin bovine serum albumin

MxSy (M = Cu, Ag, Cd)

dextran biopolymer

CdHgTe

gelatin polymer

CdxPb1−xTe

DNA, glutathione

2.9 ± 0.4 (CdSe) 9 (CdS) 14 (Cu2S) 20−50 (Ag2S) 40 (average size) ∼10.5

absorption/emission wavelength (nm/nm)

quantum yield

ref

not reported/430−530 400/500 to 800 370/not reported

not reported 2.5% not reported

91 102 106

470/not reported not reported/not reported 350/not reported centered at 400/centered at 550 260/tunable in blue region of visible spectrum 440/450 466−476/not reported absent/1100 not reported/IR luminescence not reported/1050 not reported/800−1050 500/not reported data not shown/1050−1294 not obvious/980 broad absorption peak without maximum/440 not reported/700−820

not not not not not

reported reported reported reported reported

111 120 121 122 126

not reported not reported 11.5% 1−2% 23% 3.6% not reported 1.8% not reported not reported

127 130 101 104 113 125 98 115 123 116

1.00−3.09%

128

not reported/526 ∼550/ 610 ∼520/∼560

∼30% 17.8% 17%

94 108 110

260 and ∼475−560/513 −584 ∼500−570/530−630 ∼450−550/∼500−600 450/400−650 329/497

112 124 129 132 97

380/710 not reported/not reported 340/∼500 (CdS) not obvious/∼525 (CdSe) tunable absorption/500−650

20% >40% 10% (green QDs), 30% (red QDs) not reported 15% (NH2-dendrimer) 12% (COOHdendrimer) 9% (OH-dendrimer) 11% (SA-dendrimer) not reported not reported 10−16% (CdS) 20−36% (CdSe) 15% (CdTe)

tunable absorption/500−620

60% (CdZnTe)

400/470 (CdSe) 285/340 (ZnS) not reported/470 (excitation at 410) (CdSe/ZnS) data not shown/538−616 335/420 and 590 data not shown/data not shown

not reported

131

42−50% not reported not reported

107 118 117

475/not reported (CdS)

not reported

96

624−695/580−800

not reported

93

not reported/1100−1300

not reported

109

G

92 95 114 133


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Table 2. continued materials ZnxHg1−xSe

biomimetic templates a variety of proteins (bovine serum albumin, lysozyme, trypsin, hemoglobin, and transferrin)

size distribution (nm) 3.9 ± 0.5

absorption/emission wavelength (nm/nm) data not shown/672−907

nucleation and growth of QDs.89,90 In a biomimetic system, these biomolecules and relevant analogues may serve as templates to direct and promote the growth of QDs. In particular, the biomimetic strategy takes advantage of the biophysical and biochemical properties of biomolecules (e.g., three-dimensional structure, water solubility, chelating capability, and reducing capability) to grow the QDs, and meanwhile, the biomolecules may act as capping agents to increase the solubility and stability of QDs in aqueous solution. Moreover, the biomimetic method has some unique features: (i) readily monitoring and modulating the physiochemical properties of QDs and (ii) enabling the production of QDs on a large scale. As shown in Table 2, a variety of biomimetic templates have been exploited for the synthesis of biocompatible QDs, including (i) the use of artificial cellular structures (e.g., biomimic membranes) as the synthesis templates and (ii) the use of biomolecules (e.g., nucleic acids, peptides, proteins, and enzymes) to direct the nucleation and growth of QDs. 2.2.1. Synthesis of Quantum Dots in Artificial Biomimetic Systems. The cell membrane has dual functions of (i) maintaining the stability of intracellular environments and (ii) regulating the shuttling of molecules, making it an ideal reaction compartment for nanomaterial growth. Recently, an artificial membrane with similar functions was designed to direct the growth of QDs. For example, the giant vesicles comprised of lipid bilayers can encapsulate Cd and S precursors of the CdS QDs in a defined space without the assistance of binding peptides.91 Two vesicle-based biomimetic approaches have been employed to prepare the CdS QDs: (i) one is to load the precursors of Na2S and CdCl2 individually into two vesicles followed by electric pulse-induced fusion of the two compartments to initiate the reaction, and (ii) the other is to mimic the reaction process inside the cells by incubating the vesicles that contain one precursor (e.g., Na2S or CdCl2) with an external solution containing the other precursor followed by diffusion and slow exchange of the latter precursor into the vesicles interior to initiate the reaction. In addition, amphiphilic poly(N-isopropylacrylamide)-based hydrogel has been used to prepare an artificial membrane for the encapsulation of E. coli cell extracts in a microfluidic device.92 The resulting membrane may be further used to synthesize a variety of QDs with the addition of corresponding precursors. For example, the CdSe QDs may be obtained by the addition of Cd and Se precursors (Figure 7). Notably, some biopolymers may act not only as the matrices for the QD growth but also as surface ligands to endow the QDs with different functions. For instance, gelatin solutions with different kinds of polypeptides can be used to prepare CdTe, CdSe, and NIR CdHgTe QDs with good biocompatibility.93−95 The dextran biopolymer contains abundant hydroxyls and is able to combine metal ions to produce the biopolymer shell-coated metal sulfide nanocrystals.96 The poly(amidoamine) dendrimers terminated with different groups (e.g., carboxyl, hydroxyl, and amino) can regulate both the growth and the properties of ZnTe QDs.97 Similar to the growth of nanocrystals in cells, the artificial nanopore

quantum yield 1.0−25.6%

ref 119

Figure 7. Preparation of an artificial membrane composed of hydrogel polymer and E. coli cell extracts in a microfluidic device (A) with the polymerized NIPAM monomers as an artificial membrane (B). (C, D) Synthesis of various nanoparticles with the addition of the corresponding precursors in an artificial cellular bioreactor. Figure adapted from ref 92. Copyright 2012 American Chemical Society.

building from cucurbit[7]uril can not only coordinate silver ions but also serve as the nucleation site for the growth of Ag2S QDs in the presence of sulfur precursors.98 2.2.2. Synthesis of Quantum Dots Using Biomolecules as Templates. A variety of biomolecules have been exploited as matrices for the synthesis of biocompatible QDs with desired biological functions. The nucleic acids have a unique threedimensional structure with many metal-binding groups (e.g., phosphate, hydroxyl, and nitrogen atoms) and may function as the ideal templates for QD growth.99,100 In an early study, Sargent and colleagues demonstrated the growth of infraredemitting PbS QDs on a DNA template by adding lead nitrate dropwise and subsequently injecting sodium sulfide.101 Subsequently, a number of studies have been dedicated to revealing the mechanisms of DNA-mediated QD growth.102−104 Guanosine triphosphate has been proven to be the most effective ligand for the growth of highly luminescent QDs,104,105 whereas adenosine triphosphate, cytidine triphosphate, and uridine triphosphate can only produce nonemissive solutions.104,105 Notably, the negatively charged phosphate backbone can attach metal ions through electrostatic interactions, thereby facilitating the nucleation of QDs in the presence of anion precursors (Figure 8).102 Similarly, RNAs can be employed as templates for the synthesis of QDs based on the principle that phosphate and base moieties of nucleotides H


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biological systems), which may coordinate with cadmium ions and provide a nucleation site for the growth of QDs. In particular, the coordination between pepsin and cadmium ions may induce a conformational change of pepsin from α-helix to β-sheet, facilitating the oriented growth of CdS QDs.127 In addition, the size and optical properties of CdS QDs may be tuned by simply adjusting the reaction temperature.126 The size of CdS QDs may increase from 1.7 to 5.2 nm with a gradual increase in the reaction temperature from 4 to 45 °C. Interestingly, pepsin becomes more folded at lower temperature, enabling the attachment of more pepsins to reduce the surface traps of QDs. As a result, the QDs obtained at 4 °C display the highest emission efficiency in the violet region.126 Similarly, luciferase (Luc8) can bind Pb2+ to form the Luc8Pb2+ complex, which in turn serves as a nucleation site for the growth of NIR PbS QDs in the presence of S2− (Figure 9).125

Figure 8. Growth of CdS QDs with DNA plasmid as the template. (A) The growth of CdS QDs along the DNA plasmid induces both DNA packing and GSH-mediated DNA unpacking. (B) Nanostructure of double-stranded DNA−CdS hybrid. (C) Electrostatic interaction between surface Cd2+ ions and phosphate backbones. Figure adapted from ref 102. Copyright 2011 American Chemical Society.

can direct the growth of luminescent QDs. By using either wild type or mutated E. coli tRNA as the ligand and the template, Kelley and colleagues prepared CdS QDs by simply incubating two tRNAs with Cd2+ and S2−.106 It should be noted that the modification of the phosphate backbone of DNA by thiol groups, and the replacement of the PO bond with a PS bond, may improve the binding affinity of DNA toward cations on the QD surface.105,107−111 In addition, the simultaneous modification of two phosphate domains by a nucleolintargeting motif and an mRNA-targeting motif endows the DNA−QD assembly with dual functions of targeting and imaging.108 Alternatively, the chimeric oligonucleotides with both phosphorothioate and phosphate backbone domains can not only serve as the QD ligands but also retain binding capability toward nucleic acids, proteins, and target cells.110 Moreover, with specific DNA and thrombin-binding aptamers as the ligands, the obtained DNA−QD assemblies can be used for the detection of target DNA112 and thrombin.113 Proteins and peptides are ideal templates for QD synthesis because they are ubiquitous in biological systems and contain abundant metal-binding groups, such as thiol, carboxyl, and amino. Bovine serum albumin (BSA) is the most widely used template for the preparation of various QDs, including CdE (E = S, Se),114 MxSy (M = Ag, Hg),115,116 MxSey (M = Ag, Cd, Pb, Cu),117 Mn-doped ZnS,118 and ZnxHg1−xSe QDs.119 In addition, the isolated bacterial S-layer protein,120 apoferritin,121 hemoglobin, and transferrin119 can be used as templates for the growth of QDs. As a special class of proteins, the enzymes play important roles in the growth of QDs because the enzymes can produce indispensable components, including metal-reducing components and metal-binding agents via enzymatic reactions.50 The α-chymotrypsin isolated from bovine pancreas contains five disulfide bonds, among which the Cys136− Cys201 pair can be reduced to thiols to covalently bind with the metal ions, acting as the nucleation site for the growth of QDs.122 The use of ribonuclease-A as the template may not only endow the QDs with good biocompatibility but also decrease the cytotoxicity of QDs.123,124 In addition, ribonuclease-A has excellent thermal stability, enabling simple regulation of QD emission by controlling the reaction time and temperature. Pepsin and luciferase can be employed as the matrix for the synthesis of various QDs as well.125−127 Pepsin is particularly rich in aspartic acid residues at the active site (aspartic acid plays important roles in biomineralization in

Figure 9. Schematic illustration of the preparation of NIR PbS QDs with luciferase enzyme as the template. Figure reprinted from ref 125. Copyright 2010 American Chemical Society.

Moreover, the Luc8 enzyme remains active within the Luc8PbS complex, and the bioluminescence resonance energy transfer between Luc8 and PbS QDs makes the complex emit NIR light.125 Inspired by the intracellular glutathione metabolic pathwaydirected growth of QDs, Pang and colleagues employed glutathione, the reduced nicotinamide adenine dinucleotide phosphate, and glutathione reductase to reduce Na2SeO3 (Figure 10). Meanwhile they used alanine to convert silver

Figure 10. Schematic illustration of SeO32− reducing process including (A) the reduction of SeO32− by glutathione (GSH) and (B) the reduction of GSSeSG by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione reductase (GR). Figure adapted from ref 128. Copyright 2011 American Chemical Society.

ions to Ag-alanine complexes.128 By mixing Se precursors and the Ag-alanine complexes at different molar ratios, they synthesized a series of NIR Ag2Se QDs with different sizes and emission wavelengths. Pérez-Donoso and Vásquez produced water-soluble CdTe QDs with the assistance of merely glutathione.129 Additionally, Stupp et al. designed a unique peptide-amphiphile assembly for the nucleation and I


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growth of CdS QDs.130 Myung and Chen used a bifunctional peptide to direct the growth of CdSe and ZnS through a layerby-layer deposition reaction for the preparation of core−shell CdSe/ZnS QDs.131 Ma et al. designed a dithiol peptide with dual functions of (i) monitoring the activity of target protease and (ii) templating the growth of CdTe QDs.132 This dithiol peptide may be cleaved by the target protease to yield two monothiol peptides that subsequently serve as ligands for the growth of fluorescent CdTe QDs in the presence of Cd and Te precursors.132 Xu and Lu used the arginine-glycine-aspartic acid (RGD)-containing peptides and cysteine as the coligands for the synthesis of alloyed CdZnTe QDs via the reaction of Te precursor with Cd and Zn ions.133 The obtained QDs have a high QY of 60%.

dures to transfer the hydrophobic QDs: one involves a single phase reaction between hydrophobic QD precipitates and preirradiated LA derivates in a polar solvent (e.g., methanol), and the other involves a two-phase reaction between hydrophobic QDs in a nonpolar solvent (e.g., hexane) and preirradiated LA derivates in a polar solvent (e.g., methanol). As shown in Figure 11, LA-PEG can transform into DHLAPEG under the irradiation of UV light. With the replacement of primary hydrophobic ligand (e.g., TOPO) by DHLA-PEG on

2.3. Functionalization of Chemically Synthesized Quantum Dots with Biomolecules

2.3.1. Water Solubilization of Qauntum Dots. The main challenge for the QDs synthesized in organic phase is the hydrophobic organic ligands on their surface, which limit their direct applications in biological milieu.14 Both the substitution of the original surfactant layer with water-soluble stabilizers (i.e., the ligand exchange strategy) and the encapsulation of hydrophobic QDs with amphiphilic molecules (i.e., the encapsulation strategy) can make the hydrophobic QDs eligible for biological applications.134 For the QDs to be made water-soluble, the ligand exchange strategy usually involves two consecutive processes: (i) the stripping of native surfactants (e.g., TOPO, HAD, and ODA) and (ii) replacement of the surfactants with capping ligands. The exchanged ligands may endow the QDs with good stability, appropriate size, and excellent optical properties. The bifunctional molecules with the hydrophilic groups (e.g., carboxyl group, amino group, and polyethylene glycols (PEG) group) and the metal element-binding groups are the ideal ligands for the conversion of hydrophobic QDs to hydrophilic ones. The thiol-containing molecules are the most frequently used ligands due to their abundance, extraordinary affinity, and high efficiency. Since the first experimental demonstration of mercaptoacetic acid as a permutable ligand for the generation of biocompatible ZnS/CdSe QDs by Nie and Chan,19 a series of monothiolated compounds including mercaptocarbonic acids,135−137 cysteine,138,139 and artificially thiolated agents (e.g., cyclodextrin,140 silane,141 protein,142 and DNA143) have been employed to prepare the water-soluble and biocompatible QDs. To improve the stability and the solubility of QDs, Mattoussi et al. designed a dithioled ligand of dihydrolipoic acid (DHLA) to passivate the QDs.144 To date, a variety of DHLA derivatives have been synthesized for the conversion of hydrophobic QDs to hydrophilic ones.145−151 In addition, the pyridyl-containing compounds can be used to prepare biocompatible QDs.152,153 It should be noted that the ligand exchange process usually leads to a decrease of both QD fluorescence and photostability.136,138,139,142,145,146,148,151 The widely used DHLA-based ligand exchange method requires reduction of the original lipoic acid (LA) to DHLA by chemical reducing reagents (e.g., sodium borohydride). This chemical reduction process usually suffers from a multistep reaction, difficult storage of DHLA, and inactive functional groups on the QD surface. Mattoussi et al. combined the photoligation strategy with DHLA-based ligand exchange to promote the phase transfer of hydropobic QDs to aqueous solutions.154−158 They used two photoligation-based proce-

Figure 11. Photoligation-induced ligand exchange and phase transfer of hydrophobic QDs to the water phase. (A) Two-phase reaction between TOPO-capped QDs in hexane and preirradiated LA-PEG in methanol. White light and fluorescence images of four samples with distinct colored QDs are shown. (B) Ligand exchange enables the replacement of TOPO by DHLA-PEG in the presence of tetramethylammonium hydroxide. White light and fluorescence images of four samples with distinct colored QDs are shown. (C) The final hydrophilic PEG-capped QDs. White light and fluorescence images of four samples with distinct colored QDs are shown. The samples are excited by a hand-held UV lamp at λexc = 365 nm, and QDs with maximum emission wavelengths of 540 (green), 570 (yellow), 590 (orange), and 630 (red) nm are shown. Figure adapted from ref 156. Copyright 2015 American Chemical Society. J


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the QD surface, the QDs can be easily transferred from hexane to methanol.156 After the purification and removal of organic solvents, the obtained QDs demonstrate good water solubilization. Notably, both UV and sunlight can initiate the reaction, and the addition of a small amount of tetramethylammonium hydroxide may shorten the period of ligand exchange. Recently, Mattoussi et al. performed a series of investigations to reveal the reaction process, optimize the reaction conditions, evaluate the photophysical properties of the phase-transferred QDs, and discover new photosensitive ligands.154,155,157 After the phase transfer, the QDs show good biocompatibility and can be used for in vivo imaging of mouse brain vasculature and intracellular delivery.158 Unlike the ligand-exchange strategy, the encapsulation approach usually coats the hydrophobic QDs with the amphiphilic molecules, which contain both a hydrophobic and a hydrophilic domain. The hydrophobic domain allows for the encapsulation of QDs by a hydrophobic cavity, whereas the hydrophilic domain enables the dispersion of QDs in aqueous solution. The coating molecules include silica, phospholipids, block copolymer, and liposomes. For the silica-coated QDs, the multivalency of an extensively polymerized polysilane makes the QDs water-soluble, and the easy modification of the silica surface by different groups facilitates the subsequent conjugation of QDs with biomolecules. Kotov and Rogach encapsulated the CdSe QDs with silica nanospheres (40−80 nm) and further used them as building blocks to assemble 3D colloid crystal microstructures.159 This strategy is flexible and suitable for different kinds of QDs. Rosenzweig and Chen used the organosilane-functionalized surfactant micelles to encapsulate the lipophilic CdSe QDs.160 In aqueous solution, the siloxane surfactants with long hydrophobic chains assemble into micelles spontaneously to entrap the CdSe QDs through the hydrophobic interaction. The encapsulated QDs maintain high QY, sharp photoluminescence spectra, and good stability. Notably, the encapsulation of multiple QDs into one micelle may significantly enhance the optical signals, making the QDdoped micelle a powerful probe for bioimaging applications. Bakalova et al. described a stepwise synthesis approach to prepare the single QD-based micelles by sequentially capping the QD with hydrophobic/amphiphilic/hydrophilic silica precursors. The silica-shelled single micelles not only possess high QY, small size, good monodispersity, and high transparency but also display excellent delivery capability and negligible cytotoxicity toward living cells.161,162 Recently, great efforts have been put into unraveling the encapsulation mechanisms and regulating encapsulation actions. The preincubation of hydrophobic QDs with oleic acid and stearic acid is crucial to the nucleation of the silica shell, and the preincubation of hydrophobic QDs with dodecanethiol is essential for the preservation of QD fluorescence.163 The decrease of ammonia concentration may decelerate silica formation, resulting in an ultrathin and uniform shell.164 Additionally, an ultrathin silica shell (

Toward Biocompatible Semiconductor Quantum Dots: From

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