Fluorescent/magnetic micro/nano-spheres based on quantum dots and/or magnetic nanoparticles: preparation, properties, and their applications in cancer studies

Cong-Ying Wen ^ab, Hai-Yan Xie ^ac, Zhi-Ling Zhang ^a, Ling-Ling Wu ^a, Jiao Hu ^a, Man Tang ^a, Min Wu ^a and Dai-Wen Pang *^a
aKey Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, 430072, P. R. China. E-mail: dwpang@whu.edu.cn; Fax: +0086-27-68754067; Tel: +0086-27-68756759
^bCollege of Science, China University of Petroleum (East China), Qingdao, 266555, P. R. China
^cSchool of Life Science and Technology, Beijing Institute of Technology, Beijing, 100081, P. R. China

First published on 19th January 2016

---

---

Cong-Ying Wen

Cong-Ying Wen studied at Southwest University for her BS (2008) in chemistry, and received her PhD (2014) in analytical chemistry from Wuhan University under the guidance of Prof. Dai-Wen Pang. She is now working as a lecturer at China University of Petroleum (East China). Her research interests are focused on fluorescent/magnetic nanospheres based on quantum dots and/or magnetic nanoparticles, including their construction and bioapplications.

Hai-Yan Xie

Hai-Yan Xie received a PhD in chemistry in 2004 from Wuhan University in China. Then she joined Beijing Institute of Technology and was promoted to full professor in 2010. She was awarded the Program of National Natural Science Foundation Outstanding Youth Foundation in 2014 and the Program of New Century Excellent Talents of the Ministry of Education of China in 2008. Her research interests focus on biolabeling and bionanotechnology.

Zhi-Ling Zhang

Dr Zhi-Ling Zhang received his BS (1997) in chemistry from Jilin University. He obtained his PhD (2002) in analytical chemistry from Wuhan University and finished his postdoctoral research at Ecole Normale Supérieure de Paris during 2003–2004. He is currently a professor in the College of Chemistry and Molecular Sciences of Wuhan University. His research interests are focused on microfluidics, bioelectrochemistry, nanotechnology and their application in biology and medicine.

Dai-Wen Pang

Dai-Wen Pang received his BS (1982) and PhD (1992) in chemistry from Wuhan University (WHU), and completed his postdoctoral research in virology at WHU in 1994. Then he joined WHU and was promoted to full professor in 1996. He was the Dean of the College of Chemistry and Molecular Sciences (2001–2005), and has been the Director of the Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE) from 2008 and a member of the National Steering Committee for Nanotechnology (2007-present). His interests focus on quantum dots (QDs), including live-cell synthesis and quasi-bio-synthesis of QDs, and QD-based real-time tracking of single viruses.

---

1. Introduction

Cancer is one of the most serious threats to human life. According to the World Health Organization (WHO), more than 8 million people die of cancers each year in the world, and the number of deaths increases each year.^1,2 Early diagnosis and timely effective therapy are the keys to improving the outcomes for cancer patients. However, current clinical diagnostic strategies suffer from the disadvantages of being time-consuming, having low sensitivity, requiring laborious operation, and so on;^3–5 meanwhile routinely used (chemo-) therapeutic agents for cancers are generally rapidly cleared from the circulation and usually have serious side effects.^6,7 Thus, to better ensure the safety of human life and health, developing rapid, sensitive, reliable detection methods and smart target drug delivery systems has been pursued.

In recent years, with the rapid progress of nanoscience and nanotechnology, various nanomaterials have shown great superiority and potential in the biomedicine field.^8–11 Among them, quantum dots (QDs), magnetic nanoparticles (MNPs), and functional materials based on these two, are those with the greatest application value.^12–14 QDs, as fluorescent semiconductor nanocrystals, exhibit good photochemical stability and high photoluminescent quantum yields. They have broad absorption, and a narrow and symmetric photoluminescence (PL) spectra covering a wide range (from UV to near-infrared) which can be tuned by varying the size and chemical composition of the nanocrystals, resulting in simultaneous excitation of differently colored QDs with a single wavelength.^15–17 MNPs usually refer to the nanomaterials containing iron (Fe) or cobalt (Co) as well as their alloys and oxides. MNPs become superparamagnetic at room temperature when their size is below a critical value, and such individual MNPs have a large constant magnetic moment, which behaves just like a giant paramagnetic atom with a fast magnetic response and negligible remanence (their coercivity almost equals zero).^18–20 These features avoid MNPs agglomerating at room temperature, and allow MNPs to be manipulated easily just by a magnet.

Based on such excellent properties, QDs and MNPs have shown many exciting potential applications in cancer research, such as tumor biomarker enrichment and detection, tumor cell separation and analyses, labeling and dynamic tracking, fluorescence or magnetic resonance (MR) imaging, drug delivery, and so on.^21–24 These may significantly improve our understanding of the occurrence and development of cancers, which can greatly facilitate their early diagnosis and effective therapy. Yet despite their promising potential, considerable challenges still exist in practical operation. For instance, high-quality QDs or MNPs are mainly prepared by thermal decomposition of organometallic compounds in high-boiling organic solvents, and the as-produced NPs are coated by hydrophobic ligands which are only soluble in nonpolar organic solvents.^18,25–28 For their biological application, hydrophobic NPs must be made water-soluble, and how to preserve their original optical or magnetic properties during water-solubilization treatment is a great challenge. Besides, surface engineering and biofunctionalization of NPs are also crucial for their participation in biological processes, which may encounter problems of aggregation, nonspecific adsorption, bioactivity decrease, and the difficulty to remove the excess molecules, etc.²⁵ Moreover, subsequent biological applications usually take place in very complex matrices (serum, whole blood, spinal fluid, etc.), which may significantly affect the properties of the NPs. Since QDs and MNPs were successfully synthesized, various methods for their protection and modification have been explored.^17,29–32 One of the most important methods is constructing nanoparticle-sphere (NP–sphere) composites, which combine NPs and spheres (polymer spheres, silica spheres, or similar ones in micro/nano scale) through certain interactions.^33–38 In the NP–sphere composites, the NPs are confined in closed spaces, which may protect them from the negative influence of the environment, and the spheres have hydrophilic surfaces with rich functional groups, which facilitate their modification and bioconjugation. What's more, the spheres have a large space for loading not only lots of NPs, but also drugs and other substances, which helps to construct multi-functional materials.^39–41 By regulating the type and amount of loaded NPs, encoded spheres with different fluorescence or magnetic signal can be obtained for high-throughput simultaneous analyses.^33,42–44 In addition, the NP–sphere composites also have the advantages of convenient manipulation, signal amplification, high stability, good biocompatibility, and so on. Hence, the NP–sphere composites have a very promising prospect in bioapplication, and some of them have already come into the market for scientific research or even for application in practice.

During the past decade, our group has been working on the construction and application of fluorescent/magnetic biotargeting nanospheres based on QDs and/or MNPs. We have made a series of achievements and accumulated some experiences and perspectives.^34,45–60 Herein, we mainly review the four currently used methods for preparing QD/MNP-based fluorescent/magnetic micro/nano-spheres, and then summarize the characteristics of the achieved NP–sphere composites. Furthermore, we discuss their recent applications in cancer research in detail, ranging from separation, diagnosis, to therapy. Finally, we conclude with an outlook for the challenges and future directions of the NP–sphere composites in reaching clinical applications.

2. Construction of the fluorescent/magnetic micro/nano-spheres

Based on nanoparticles (NPs, including QDs and MNPs), fluorescent/magnetic micro/nano-spheres are constructed mainly through four methods (Scheme 1): (I) embedding of NPs into micro/nano-spheres; (II) incorporation of NPs during the micro/nano-sphere formation process; (III) assembly of NPs onto the surfaces of micro/nano-spheres; (IV) in situ synthesis of NPs within the pores of micro/nano-spheres. In techniques (I) and (III), micro/nano-spheres and NPs are pre-prepared and then combined together. In the other two techniques, combination occurs during the formation process of micro/nano-spheres (II) or NPs (IV). The following provides a brief overview of the principles and characteristics of the four methods.

2.1. Embedding NPs into the micro/nano-spheres

In this method, NPs are embedded into micro/nano-spheres through the interactions (including hydrophobic interaction,^33–35 electrostatic interaction,^61,62 and so on) between NPs and the pores of the spheres (Scheme 1(I)). Nie's group³³ first embedded hydrophobic QDs into polymer spheres for optical coding (Fig. 1A), in which a solvent mixture containing chloroform and propanol or butanol (5:95 by volume) was used to swell the hydrophobic pores of the microspheres to facilitate the transport of the QDs into the microsphere interior by hydrophobic interactions. Then the achieved QD-embedded microspheres were dispersed in a polar solvent and the pores shrank, allowing the QDs to be retained. Based on similar principles, our group,^34,45,46 for the first time, simultaneously incorporated QDs and iron oxide nanocrystals into much smaller polymer nanospheres (Fig. 1B, diameter: ca. 200 nm). With subsequent biofunctionalization, we obtained fluorescent-magnetic-biotargeting trifunctional nanospheres. Not only had the nanospheres attained the integration of multiple functionalities, but also their nanoscale gave them higher reaction flexibility and faster binding kinetics in biological applications. We applied for a patent on this method in 2003.⁶³ Afterwards, Nie's group³⁵ also embedded QDs and MNPs into mesoporous silica microspheres to fabricate dual-functional carriers for optical encoding and magnetic separation (Fig. 1C). The embedding method is one of the most convenient methods for the fabrication of fluorescent/magnetic micro/nano-spheres. What's more, high-quality oil NPs are directly incorporated into the hydrophobic pores of the spheres, and the organic ligands on the NP surface are hardly damaged at all, allowing the NPs to maintain their optical or magnetic properties to the greatest extent.²⁹ However, the embedding process has a certain randomness, and the distribution of NPs through the obtained fluorescent/magnetic sphere interior is not always uniform.^64,65 The NPs are mainly located in the outer zones of the spheres,³³ which limits their loading capacity. Another disadvantage is that the NPs may leak out of the sphere pores in nonpolar suspending media.

2.2. Incorporating NPs during the micro/nano-sphere formation process

Scheme 1(II) briefly shows this fabrication process: NPs are incorporated into the micro/nano-sphere interior as the monomers are polymerized into spheres, mainly by suspension,^65–67 dispersion,^68,69 emulsion,^70–72 or condensation polymerization.^73,74 Zhang et al.⁷⁵ (Fig. 2A) utilized polymerizable surfactants to transfer aqueous CdTe nanocrystals into a solution of styrene monomer, and through subsequent radical polymerization, they got CdTe-PS composites. Meanwhile oil QDs could be directly encapsulated into polystyrene spheres induced by a miniemulsion polymerization approach (Fig. 2B),⁷⁶ and further a method for kinetically entrapping QDs by cross-linking the polymer was developed, which helped to maintain a uniform dispersion (Fig. 2C).⁶⁵ Magnetic spheres can also be constructed in similar ways. Li et al.⁷³ (Fig. 2D) prepared magnetic microspheres through the polymerization of urea and formaldehyde, which induced Fe₃O₄ nanoparticles to aggregate in urea-formaldehyde (UF) resin, followed by silanization and calcination. The obtained magnetic microspheres had a high saturation magnetization (61.38 emu g⁻¹), which was attributed to the complete removal of UF resin by calcination to achieve high magnetic nanoparticle content. Recently, microfluidics have been applied to the fabrication of NP-polymer composites using droplet-assisted polymerization, which have shown great advantages of precise control, convenient manipulation, and fast reaction kinetics (Fig. 2E).^77–81 In these polymerization methods, NPs extend throughout the sphere interior during sphere formation, which improves the NP distribution inhomogeneity problem to a certain extent, and efficiently avoids NP leakage. But the reaction conditions usually need to be carefully controlled, and the NPs may aggregate during polymerization, which may influence their fluorescence or magnetic properties.^65,82,83 Besides, this method also faces the limitation of NP load capacity and the difficulty to control precisely the number of loaded NPs, which is not very suitable for multiple encodings.

2.3. Assembling NPs onto the surfaces of the micro/nano-spheres

With layer-by-layer assembly methods (Scheme 1(III)), NPs can be decorated onto the surfaces of micro/nano-spheres through the interactions between the NPs and the moieties on the spheres, such as electrostatic interaction,^84–86 coordination interaction,³⁸ bioaffinity,^87,88 disulfide linkage,⁸⁹ and so on. Compared with the first two methods, assembly of NPs on a sphere surface doesn't need to consider the NP size or shape. The assembly usually happens in a quasi-homogeneous system, and the reaction conditions are very mild, usually gentle shaking at room temperature. Wilson et al.⁸⁴ (Fig. 3A) successfully attached negatively charged CdSe/ZnS QDs on the positively charged surfaces of magnetic microspheres which were previously coated with a foundation layer of poly(allylamine). QD deposition made the ξ-potential of the spheres more negative, and then through further assembly of another positive polymer (poly(ethyleneimine), PEI), they could attach a second layer of QDs, followed by coating three and a half bilayers of PEI + poly(sodium 4-styrenesulfonate) (PSS). By repeating the coating procedures (steps 2 and 3 in Fig. 3A), multiple layers of QDs could be located in a densely packed dark shell surrounding the microspheres. Even after assembling 15 layers of QDs, the microspheres still had well-defined core–shell structures, suggesting that additional layers could be assembled without disruption. The multilayer assembly technique facilitated the high NP load capacity of this method. What's more, the photoluminescence intensity increased uniformly in proportion to the number of QDs assembled, which demonstrated that the assembly imposed a high degree of control. Thus, it's conceivable that by tuning the coating amount and the type of NPs in each layer as well as changing the assembly layers, encoding can be achieved precisely and the code number can be greatly expanded. Wilson et al.⁹⁰ then improved their method, and directly deposited hydrophobic QDs on PEI coated microspheres through forming covalent bonds between PEI and the QDs. Compared with electrostatic or covalent interaction, biological self-assembly has now becomes more promising and attractive owing to its high and specific affinity. Rauf et al.⁸⁷ (Fig. 3B) produced QD barcodes by the layer-by-layer assembly of streptavidin- and biotin-functionalized QDs. The streptavidin–biotin interaction was sufficiently strong to stabilize the assembled codes, and they provided a reagentless method to bring modular components together. Meanwhile, the surface-bound streptavidin on the resultant spheres enabled them to conjugate with biological molecules without further functionalization, and also made them minimally non-specific. However, the assembly strategies mentioned above have usually been applied to microspheres rather than nanospheres. While spheres on the nanoscale have minor steric hindrance and higher flexibility in bioapplications, their fabrication is very important. Therefore, our group³⁸ expanded the assembly method to nanospheres (Fig. 3C) in which not only hydrophobic QDs but also hydrophobic MNPs could be straightforwardly assembled on the surface of nanospheres through coordination between the primary amines of PEI and metallic atoms from NPs. Hence, with this method, fluorescent encoded, magnetic encoded, and fluorescent-magnetic dual-encoded nanospheres were all precisely constructed. We further calculated its huge encoding potential, which would surely increase with the number of coating layers. From the above review, it can be concluded that the assembly method gains the advantages of high load capacity, high controllability, good reproducibility, mild conditions, and can utilize various kinds of interactions to combine NPs and spheres without consideration of the NP size or shape. Nevertheless, it also can be seen that the assembly process is very tedious and time-consuming. Some assembly methods even undergo a transfer from water into the oil phase, then from the oil into the water phase, over and over again. Besides, another outer shell is usually needed to cap the obtained fluorescent/magnetic spheres for protection and conjugation.^53,84,90

2.4. Synthesizing NPs in the micro/nano-spheres

When the porous microspheres are saturated in a solution containing precursors for synthesizing QDs or MNPs, by controlling certain conditions, the precursors can directly react to form NPs within the pores of microspheres, thereby providing the microspheres with fluorescence or magnetic functionality (Scheme 1(IV)). As early as the 1980s, Ugelstad et al.^91,92 used this method to pioneer studies on the fabrication of magnetic polymer microspheres (Fig. 4A). The porous polymer microspheres were first impregnated in solutions of Fe(II) and Fe(III) salts, then treated with a base to synthesize MNPs, finally, polymerization was carried out on the surface of the magnetic microspheres to fix the iron oxides and supply functional groups. Ugelstad et al. applied for several patents, and successfully commercialized the magnetic microspheres, which were widely used by researchers, under the name “Dynabead®”.⁹³ Afterwards, in situ synthesis of QDs into porous microspheres was also developed by Yang et al.⁹⁴ As Fig. 4B shows, the polymer microspheres were first swollen in chloroform containing Cd, Zn, Se, and S precursors which diffused into the microspheres subsequently. Then, with thermal decomposition, QDs were synthesized into microspheres, and the polymer spheres were de-swelled gradually as the chloroform evaporated. By changing the precursor ratios, they obtained fluorescent microspheres with a wide range of emission wavelengths (Fig. 4C). Directly synthesizing NPs within the pores results in a very uniform distribution of the nanocrystals. The mesopores on the sphere surface are sealed after preparation, which causes the NPs to be fixed into the microspheres and efficiently avoids NP leakage. But this method was mainly applied to micro-scale spheres, and it is difficult to fabricate a variety of barcodes or fluorescent-magnetic bifunctional spheres, since only one kind of NP can be fabricated under certain conditions.

Above we have given a general review of the four methods for fabricating NP–sphere composites, mainly from their design principles, operation processes, and their characteristics. Each method has its own advantages and limitations, which are summarized in Table 1. Researchers, on the one hand, have always been trying to improve these methods, and some disadvantages even have been overcome under certain conditions. On the other hand, researchers can select an appropriate method according to their application demands and the experimental conditions available. They can even couple two or more methods to achieve better effects. For example, the combination of embedding and assembly can maximize space utilization, which may help to construct much smaller spheres with high brightness or a fast magnetic response, thus efficiently solving the contradiction between the size and signal intensity.

---

3. Properties of the fluorescent/magnetic micro/nano-spheres

First of all, the fluorescent/magnetic micro/nano-spheres successfully inherit the unique fluorescence or magnetic properties of the QDs or MNPs. Meanwhile, compared with the NPs alone, the fluorescent/magnetic micro/nano-spheres show additional advantages in biological applications, which are discussed in detail below.

(1) Fluorescent/magnetic micro/nano-spheres have much higher stability, and the optical and magnetic properties of NPs can be maintained very well even in harsh conditions. For one thing, during the incorporation process, the ligands on the NP surface are hardly damaged, allowing the NPs to maintain their characteristics to a great extent. For another thing, after incorporation, the NPs are confined in enclosed spaces, which may protect them from the negative influence of the environment. Nie's group³³ reported that, unlike free QDs in aqueous buffer, the embedded QDs were stable under temperature cycling conditions, and Yang et al.⁹⁴ also reported that the fluorescence intensity of their QD barcodes could be maintained effectively for at least a month. Meanwhile, our group had done some experiments to monitor the stability of the NP–sphere composites. The fluorescent nanospheres (FNs) fabricated by our embedding method^34,45–47 were dispersed into pure fetal bovine serum, and their fluorescence intensity changed little during 30 min incubation (Fig. 5A). Besides, our previous studies⁵³ had shown that even in whole blood the magnetic nanospheres (MNs) made by our developed assembly method could remain monodisperse (Fig. 5B) and be re-collected at nearly 100% with only a commercial magnetic scaffold. Furthermore, we also monitored the stability of six batches of FNs and MNs with storage time, which were simply dispersed in pure water and stored at 4 °C, noting their fluorescence and magnetic properties, dispersibility, and usability. As shown in Fig. 5C and D, the fluorescence intensities of the six batches of FNs were essentially unchanged with increasing storage time (mean relative standard deviation (RSD): 10.0%), and the fluorescence spectra of the FNs after 12 month storage remained almost the same with the newly prepared FNs. The magnetic properties also exhibited high stability. In 12 months, the saturation magnetizations of the six batches of MNs hardly changed (mean RSD: 5.1%), and the MNs retained superparamagnetic properties at room temperature (Fig. 5E and F). Fig. 6 showed that the hydrodynamic sizes of FNs (Fig. 6A) and MNs (Fig. 6C) had no obvious increase trend with the storage time, and their polydispersity index (PDI) values remained at about 0.1 (Fig. 6B and D), which confirmed that the FNs and MNs didn't aggregate and retained good monodispersibility for at least 12 months. What's more, after one year storage, the FNs and MNs were modified with the anti-epithelial-cell-adhesion-molecule (anti-EpCAM) antibody to fabricate immunofluorescent nanospheres (IFNs) and immunomagnetic nanospheres (IMNs), which were used, respectively, to label and capture breast cancer cells. (The related experimental methods are in our previous work.^51,53) From Fig. 7, it can be seen that the anti-EpCAM IFNs successfully labeled SK-BR-3 cells with red fluorescence, and the anti-EpCAM IMNs efficiently captured more than 95% of MCF-7 cells, while the FNs and MNs showed low nonspecific adsorption. All of the above long-term monitoring convincingly demonstrates that the fluorescent/magnetic spheres have high stability, which makes them excellent fluorescence probes and magnetic separation tools. Another merit of protection of NPs into micro/nano-spheres can be exhibited by their modification. Modification, especially the covalent conjugation and coordination conjugation, usually leads to NP aggregation and reduction of brightness and stability of the resultant conjugates,²⁵ which can be almost completely avoided in sphere modification.

(2) Micro/nano-spheres have a large space for loading. Firstly, a great number of NPs can be encapsulated into a single sphere, which can provide a highly amplified signal and improve the sensitivity in bioassay. As Nie's group³³ reported, a single 1.2 μm microsphere was able to be incorporated with thousands of QDs. Our group also calculated that about 1800 QDs were attached on one layer of a 250 nm nanosphere by the assembly method.⁵⁴ As we know, MNPs usually show a slow magnetic response, and need to be captured under a high-gradient magnetic field, which limits their application. For instance, the magnetic activated cell sorting technology developed by Miltenyi Biotech Corp.,⁹⁵ known as MACS-technology, utilizes 50 nm MNPs to capture target cells with the help of a high-gradient magnetic separation-column that aren't available generally. The collection of MNPs into a single sphere can facilitate a much more rapid magnetic response and cause separation by an ordinary magnetic scaffold to be achieved within only seconds to minutes (Fig. 8A and B).^49,53,96 Thus, compared with the monodisperse MNP colloids, magnetic spheres show better isolation performance, which extends the magnetic capture to common laboratories, and even to on-site application. Secondly, the micro/nano-spheres can simultaneously load QDs, MNPs, drugs, and so on, to fabricate multi-functional materials with fluorescence tracking, magnetic separation, diagnosis, therapy, and other functionalities.^25,97–99 Multi-functional materials have great potential applications in cancer study, which will be discussed in part 4. Thirdly, as we mentioned repeatedly in part 2, by regulating the kinds and amounts of NPs loaded, we can obtain encoded devices with different fluorescence or magnetic signals (Fig. 1A, 3C and 4C), which have become an important barcoding technology to supply a platform for high-throughput simultaneous analyses.^33,38,94

(3) Micro/nano-spheres supply large surfaces that can be utilized, and the ones with hydrophilic surfaces are usually chosen in order to be useful in biological applications. For the widely used high-quality QDs and MNPs, which are mostly produced by organic phase synthesis, incorporation with hydrophilic spheres is an efficient method for water solubilization. What's more, the micro/nano-spheres have rich groups on their surfaces^25,32 which greatly facilitate subsequent bio-functionalization and enable the spheres to be coupled with many more biomolecules, greatly improving the binding efficiency in the bio-recognition. As we have calculated, the number of the active affinity sites on each antibody-modified nanosphere (∼380 nm) was about 100,⁵³ and the active streptavidin number on each streptavidin-conjugated nanosphere (∼250 nm) could reach 200 (unpublished data). On the other hand, these groups also can be further modified to obtain other reactive groups (such as –COOH, –NH₂, –SH, –COCl, etc.), which can meet various experimental requirements, or they can be conjugated with certain functional molecules to improve their biocompatibility or reduce their non-specific adsorption. For example, many researchers have already introduced PEG to the sphere surface to reduce steric hindrance and prevent non-specific binding.^{29,51,100,101}

(4) Compared with NPs, the micro/nano-spheres can be more conveniently manipulated due to their larger size. In the modification and bio-functionalization of QDs, ultrafiltration, chromatography, gel electrophoresis, or a similar separation technology is usually used to remove the excess molecules, which makes the modification process tedious and time-consuming.¹⁰² Nonetheless, these manipulations still cannot achieve efficient and complete removal, and the residual free molecules may influence the bio-recognition process. Relatively speaking, the modification of micro/nano-spheres is much more convenient and time-saving, usually by common centrifugation or magnetic separation.^45,54

Of course, the fluorescent/magnetic micro/nano-spheres also have some disadvantages, and the greatest one is that their reaction kinetics and flexibility become slower as their size increases. Fabrication of high bright fluorescent spheres or quick-response magnetic spheres with a much smaller size is one of the goals that researchers have always pursued. However, despite their defects, fluorescent/magnetic micro/nano-spheres have been proved to have promising applications in biological research.

4. Fluorescent/magnetic micro/nano-spheres for cancer study

This section summarizes the biological applications of fluorescent/magnetic micro/nano-spheres, mainly in the field of cancer research (Fig. 9).

4.1. Separation and enrichment

In bioassays, as targets usually exist within complex biological matrices, their highly efficient isolation and enrichment are crucial and necessary steps for accurate analyses. As mentioned above, magnetic spheres have excellent superparamagnetic properties at room temperature, which enables them to be conveniently manipulated just by an external magnet. Besides, they have a high surface-to-volume ratio and fast kinetics in solution, and their bio-modification is easy. Therefore, the magnetic sphere-based separation technology has excellent isolation performance and has been widely used in bioassays. The isolated objects can be from ions,^103,104 small molecules^105,106 or biomacromolecules,^{49,54,107,108} to viruses,^52,56–58 bacteria^51,59,109 or cells,^110–112 and so on. In cancer study, magnetic spheres are mainly applied to the isolation of tumor cells, tumor biomarkers, and other substances relative to the occurrence, development, diagnosis, and treatment of cancer.

Tumor cell isolation is one of the earliest applications of the magnetic spheres, and our group has done a series of work in this field. As early as 2005, we had successfully used folic acid modified fluorescent-magnetic nanospheres to capture Hela and MCF-7 cancer cells.³⁴ Then we further constructed wheat germ agglutinin-conjugated fluorescent-magnetic trifunctional nanospheres (WGA-TFNS) to recognize human prostate carcinoma DU-145 cells with little cytotoxicity, and by only 0.33 mg of WGA-TFNS, more than 1.0 × 10⁵ DU-145 cells were able to be captured.⁴⁵ The experimental manipulation is very convenient. Just as Fig. 10A showed, bio-targeting nanospheres were first added to the matrix, followed by gentle incubation, and then a magnet was used to capture the cells bound with the nanobioprobes. In our subsequent work,⁵⁰ by embedding QDs of different colors red fluorescent magnetic nanospheres and green fluorescent magnetic nanospheres were fabricated, and they were then respectively modified with two kinds of antibodies to recognize and isolate multiple types of tumor cells with high efficiency and selectivity. What's notable in this work is that rapid separation of a small number of spiked tumor cells in a large population of cultured normal cells (about 0.01% were tumor cells) was achieved simply and inexpensively without any pretreatment before cell analysis. It can be seen that magnetic spheres have the ability of highly efficient isolation and enrichment, and therefore they have recently been applied to the research of circulating tumor cells (CTCs).^{110,112–114} CTCs are tumor cells which are shed from tumors into the bloodstream, and their detection and investigation is of great significance to early diagnosis of tumors, treatment monitoring, prognosis, and metastasis diagnosis.^115–118 However, CTCs are extremely rare in the extremely complex matrix (only up to hundreds of CTCs out of >10⁹ hematological cells in 1 mL of blood), which makes their investigation extremely difficult.^119–121 Magnetic separation technology facilitates the efficient purification and enrichment of CTCs and greatly promotes CTC study. An automated immunomagnetic enrichment technology for CTC detection has been approved by the US Food and Drug Administration (FDA) and comes into the market, known as CellSearch, which shows high accuracy and sensitivity with good reproducibility.^122–124 In the CellSearch system,¹²⁵ IMNs (120–200 nm) modified with anti-EpCAM antibody and biotin analogue were first used to capture CTCs, and then streptavidin is added to bind the biotin analogue, followed by the treatment of excess IMNs, which can bind to the bound streptavidin to increase the number of IMNs combined with CTCs, hence achieving a fast magnetic response. After magnetic separation, biotin is added to bind competitively streptavidin to release excess IMNs. Based on a similar principle, Lu et al.¹²⁶ developed a biotin-triggered decomposable immunomagnetic system for the capture and release of CTCs. As shown in Fig. 10B, the immunomagnetic beads, which were modified with antibodies through the interaction between Strep-Tactin and Strep-tag II, were first used to recognize and capture tumor cells, and then D-biotin was added to compete effectively with Strep-tag II for its higher affinity with Strep-Tactin. As a result, the Strep-tag II-tagged antibody was detached from the immunomagnetic beads, and the captured tumor cells were easily released. Quantitative experiments showed that 70% of the captured cells could be released, and 85% of the released cells remained viable. However, these methods introduced more steps before tumor cell analyses, which not only made the operation procedure more complicated, but also increased cell loss and influenced cell viability. Thus, we⁵³ used a layer-by-layer assembly method to construct quick-response MNs, nearly all of which could be captured by 1 min attraction with a commercial magnetic scaffold. In addition, their nanometer scale provided them them fast kinetics and good biocompatibility. After modification with the anti-EpCAM antibody, the obtained IMNs were able to successfully capture tumor cells in whole blood with an efficiency of more than 94% via only 5 min incubation. Moreover, the IMN binding had little influence on the isolated cells, and the cells remained viable at more than 90.5%, which could be directly used for culture, reverse transcription polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification without disassociating IMNs (Fig. 10C). We further successfully applied the IMNs to the detection of CTCs in cancer patient peripheral blood samples, showing great potential in CTC studies.

In clinical cancer diagnosis, compared with tumor cells, biomolecule biomarkers such as proteins, genes, and so on, are much more widely used.^4,127,128 In their detection, magnetic separation is also a preferred choice for pre-concentration. The target biomarkers can be isolated from the complex matrix into a relatively pure environment with a highly increased concentration, which can significantly improve the anti-interference ability and the sensitivity of the detection method. Moreover, magnetic separation can be conveniently coupled with various identification techniques, such as fluorescence observation, electrochemical detection, polymerase chain reaction (PCR), culture, and so on.^43,129–132 The sandwich-type detection method is a typical design, in which bio-modified magnetic spheres are used to capture the targets, followed with the addition of bio-modified signal probes to form sandwich complexes. Through monitoring their signal, qualitative detection or quantitative analysis can be achieved. Li et al.¹³¹ developed an immune sandwich assay for carcinoembryonic antigen (CEA) detection by coupling upconversion phosphors (UCPs) and magnetic beads (MBs). As shown in Fig. 11A, UCPs conjugated with the anti-CEA antibody were used as reporter probes, and magnetic beads modified with another biotin-tagged anti-CEA antibody were employed as separation tools. With the assistance of a magnet, the as-formed immune sandwich could be readily isolated from the assay matrix for sensitive detection. Gu's group¹³³ reported a phage-mediated method to count cancer-biomarker miRNA molecules at attomolar concentrations just by the naked eye. In this method (Fig. 11B), miRNA-capturing magnetic microparticles were used to capture target miRNA which was simultaneously combined with the phage-gold nanoparticle couple (in a one-in-one manner) modified by another capturing oligonucleotide. After magnetic separation, the phage was released from the resultant sandwich complex containing equimolar phage and miRNA, and developed into one macroscopic fluorescent plaque in a Petri dish. By counting the plaques with the naked eye, they achieved the quantification of miRNAs with ultrasensitivity. In these methods, without the help of MNs, high sensitivity and strong anti-interference ability would not have been attained. Recently, magnetic sphere-based separation technology has been integrated with microfluidics to avoid the drawbacks involved in conventional magnetic separation methods which are performed in Eppendorf tubes with significant reagent consumption and a tedious washing process.^57,134,135 Our group⁵ used a nickel pattern to generate high magnetic field gradients for the formation of controllable superparamagnetic bead (SPMB) patterns in microfluidic channels. CEA and alpha-fetal protein (AFP) could be simultaneously captured respectively in two channels, and then with high luminescent QDs as fluorescence indicators, sensitive and rapid detection of dual cancer biomarkers was achieved directly in serum (Fig. 12).

With the development of magnetic separation techniques, they are no longer limited to scientific research, but have been extended to practical applications. Corporations, such as Invitrogen, Bangs, Ademtech, and so on, have fabricated a variety of magnetic spheres with different functionalities to meet the needs of researchers and technical staff. For example, Dynabeads of various sizes can be modified with different ligands (such as antibody, protein, antigen, DNA/RNA, etc.), and are available for many applications.¹³⁶

4.2. Cancer diagnosis

On the 2013 World Cancer Day, the UICC (Union for International Cancer Control) indicated that, advances in understanding risk and prevention, early detection and treatment have revolutionised the management of cancer leading to improved outcomes for patients, and in addition, investing in prevention and early detection of cancer is cheaper than dealing with the consequences.¹³⁷ Thus, early diagnosis of tumors has great medical significance. Recently, cancer is diagnosed mainly through medical imaging and tumor biomarker monitoring, in which QDs, MNPs and the materials based on them show great application advantages due to their unique optical and magnetic properties.^12,138–141

During the past few decades, scientific breakthroughs from physics, chemistry, engineering, and medicine have led to the rapid development of biomedical imaging techniques, such as magnetic resonance imaging (MRI), computer tomography (CT), positron emission tomography (PET), optical fluorescence imaging, single-photon emission computed tomography (SPECT), photo-acoustic (PA) imaging, ultrasound (US) imaging, and so on.¹⁴² Among them, MRI and optical fluorescence imaging, which are noninvasive and avoid using harmful radiation, show the most value in medical applications.^143–145 QDs, compared with traditional fluorescent dyes, are much brighter and have improved photostability, which enable long-term observation and imaging. Moreover, QDs have large Stokes shifts and broad excitation spectra, facilitating multiplexed in vivo cell detection and tracking.¹⁵ Nie's group^12,25 compared the imaging sensitivity of QDs and green fluorescent protein (GFP). Just as Fig. 13A (a–c) shows, although the QD-tagged cells (Fig. 13A (b)) and the GFP-transfected cells (Fig. 13A (c)) were similarly bright in cell cultures, only the QD signal was observed in vivo. Further, with a single light source, they explored multicolor imaging with QD-encoded microbeads (Fig. 13A (d)). It is worth mentioning that micro/nano-composites based on near-infrared (NIR) QDs have recently attracted more-and-more attention in the in vivo imaging field, owing to the advantage of NIR fluorescence in terms of its large penetration depth while the tissues (or cells) emit low auto-fluorescence in this region; moreover the NIR QDs can avoid the use of Cd, Pb, or similar toxic precursors, which are more suitable for in vivo imaging.^146,147 Our group had successfully prepared Ag₂S and Ag₂Se QDs for NIR fluorescence imaging in vivo.^148–150 While compared with optical imaging and other imaging techniques, MRI usually has much higher spatial resolution. MRI has now become an essential part of modern clinical imaging, in which magnetic nanocomposites have been developed as a novel excellent MRI contrast agents.^138,143 Magnetic nanocomposites have high magnetic signal strength, greater contrast enhancement and relatively low cytotoxicity. What's more, their properties such as magnetism, size, and facile modification can be tuned conveniently to meet different imaging demands.^138,142,144 Xie et al.¹⁵¹ prepared 4-methylcatechol coated MNPs, which could be directly conjugated with a peptide, c(RGDyK), via the Mannich reaction. When administrated intravenously, these MNPs accumulated in tumor cells, which were readily tracked by MRI. As shown in Fig. 13B, the MR signal intensity of tumor changed significantly after the injection of MNPs. However, MRI also has its limitations, such as its sensitivity still cannot compare with that of PET which can reach up to the femtomolar level of the biological targets of interest.^138,142 Thus, multimodal imaging, which can integrate the complementary merits of different imaging modalities to enhance both the sensitivity and accuracy of clinical imaging diagnostics, is a very important development trend.^145,152,153 For example, the combination of MRI and PET can achieve the high resolution of MRI and the high sensitivity of PET, which provides more detailed and accurate imaging information than using each alone.¹⁵⁴ Magnetic nanocomposites can carry a wide range of other imaging moieties (fluorescent molecules, QDs, and radioisotopes etc.) either inside the composites or on their surfaces (Fig. 13C),¹⁴³ which makes them an important platform for multi-modal imaging applications. Shin et al.¹⁴² summarized various multi-modal imaging techniques in great detail, as shown in Fig. 13D, including T₁–T₂ dual mode MRI,¹⁵⁵ MRI-optical dual mode,^156,157 MRI-PET/SPECT dual mode,¹⁵⁸ MRI and US imaging,¹⁵⁹ MRI and photoacoustic (PA) imaging,^160,161 and so on. Both Park et al.¹⁶² and Shibu et al.¹⁶³ successfully fabricated MNP-QD hybrid nanocomposites to obtain a bimodal contrast agent for combined MRI and fluorescence imaging. The in vivo MR and fluorescence images showed magnetic and fluorescence contrast enhancements, which confirmed the advantages of the combination of MRI (for the determination of full anatomical distribution in vivo) and optical imaging (for microscopic resolution and in vivo fluorescence imaging). Apart from QDs, NIR-to-visible upconversion nanoparticles (UCNPs) are also utilized as MR-optical imaging probes, due to their narrow emission peak, high photostability, deeper penetration depth, and the use of NIR light as an excitation source.¹⁶⁴ Besides, fluorescent molecules, radioisotopes, gold nanoparticles, and other kinds of imaging agents could also be coupled with MNPs to achieve multimodal imaging.^142,165,166 In summary, a detailed explanation of the features of the bioimaging techniques with QD/MNP-based nanocomposites is supplied in Table 2. From all the above, it can be seen that QD/MNP-based nanocomposites, as the new generation of biomedical imaging agents, will greatly benefit accurate cancer detection.

---

As for tumor biomarker detection, micro/nano-composites based on QDs or MNPs can be directly employed as reporter probes for qualitative or quantitative analysis of targets.^139,141 Since hundreds of NPs can be encapsulated into a single sphere, which can provide a highly amplified and stable signal,^51,189 a higher sensitivity is usually achieved. Zhou et al.⁷⁶ used QD-based fluorescent nanospheres to perform fluorescent lateral flow immunoassay (LFIA) strips for highly sensitive and rapid detection of AFP, and the detection limit could reach 0.1 ng mL⁻¹, a sensitivity 200 times higher than that of commercial colloidal gold-labeled LFIA strips. Our group⁵¹ also utilized fluorescent nanospheres coupled with magnetic nanospheres to develop a convenient one-step strategy for detecting bacteria or cancer cells, which showed a lower loss and higher sensitivity than the traditional two-step method (as low as 10 cells per mL can be efficiently captured and identified). Attributed to the high stability of the nanocomposites, this strategy had a strong anti-interference ability, which can be directly applied to serum samples, or even whole blood samples. More notably, high throughput multiplexed detection could be achieved with QD-encoded spheres. The basic principle is shown in Fig. 14A, and the target of interest is identified by the barcode signal and quantified by the label signal.¹⁹⁰ Early in 2001, Nie's group³³ had successfully fabricated QD-tagged microbeads for multiplexed detection of DNAs (Fig. 14B) using spectral analysis, and up to now, biomarker proteins, cancer cells, etc. all have been simultaneously identified, indicative of their great potential in clinical applications for cancer detection.^50,191–194 Also, other researchers distinguished several fluorophores by their life time for multiplexed measurements, which can help in the event of too much spectral overlap.^195,196 Apart from utilizing fluorescence signals, magnetic composites can also be excellent reporter probes, which usually lead to ultrasensitive detection, mainly because most biological samples exhibit virtually no magnetic background. Tan's group¹⁴¹ successfully used aptamer-conjugated magnetic nanoparticle (ACMNP)-based nanosensors for pattern recognition of cancer cells by magnetic relaxation measurements. As shown in Fig. 14C (a), when multiple magnetic nanosensors bound to their target cells, they tended to aggregate to form clusters, inducing coupling of the magnetic spin moment. This generated strong local magnetic fields, leading to inhomogeneities that accelerated the spin-dephasing of the adjacent water protons and resulted in a decreased spin–spin relaxation time (T₂). With this method, the magnetic nanosensors were able to detect as few as 10 cancer cells in 250 μL of sample with high selectivity and sensitivity. Furthermore, as the number of receptors increased on the cells, ΔT₂ became larger (Fig. 14C (b)), based on which cancer cell types with different expression levels of membrane receptors could be successfully differentiated. Besides, some researchers also developed a detection system utilizing the giant magnetoresistance (GMR) of magnetic nanocomposites, which can reach zeptomole or even single-molecule sensitivity.^197–199

4.3. Cancer therapy

For cancer patients, timely and effective treatment is another key to improve their survival. However, routinely used (chemo-) therapeutic agents usually suffer from some serious disadvantages. For instance, their low molecular weight generally causes them to be cleared rapidly from the circulation. Their small size and/or their high hydrophobicity make them present with a large volume of distribution. Consequently they don't accumulate well at the pathological site, and what is more serious is that their accumulation in healthy tissues may cause inadvertent side effects.^7,200,201 To solve these problems, various kinds of drug delivery systems have been designed to: (1) maintain the drug concentration at safe levels; (2) achieve appropriate target site accumulation; and (3) engineer a long circulation to achieve longer half-life. The drug delivery system can perfectly balance the efficacy and the toxicity of anticancer drugs, which can greatly improve therapeutic efficacy.

In recent years, the progresses in bio-nanotechnology and nanomedicine have brought new opportunities to drug delivery systems for cancer therapy.^6,11,202 Just as described in Part 3, micro/nano-composites have a large space for loading QDs, MNPs, drugs, and so on, which enables them to be an excellent platform for engineering multifunctional devices. Among them, the magnetic targeted drug delivery systems have drawn special attention due to the unique magnetic properties of MNPs. For instance, through magnetic guidance, composites with drugs and MNPs can be delivered to lesion sites via applying an external magnetic field, and then release drugs under some stimulus (pH, field, heat, etc.), which can greatly reduce toxic side effects.^203–206 Thomas et al.²⁰⁷ incorporated zinc-doped iron oxide nanocrystals (ZnNCs) into mesoporous silica nanoparticles. Then the base of the molecular machine was attached to the nanoparticle surface, followed by drug loading and capping (Fig. 15A). When the drug delivery system was set to an alternating current (AC) magnetic field, the nanocrystal generated local internal heating to cause the molecular machines disassemble and allow the drugs to be released. Thomas et al. then used the delivery system to treat breast cancer cells, and 37% of the cells were killed, suggesting that this system would be a promising tool for target cancer therapy. Now, magnetic targeted drug delivery systems have been applied to in vivo research, and have also achieved good therapeutic efficacy. Li et al.²⁰⁸ developed a magnetic mesoporous silica nanoparticle-based PEI and fusogenic peptide-functionalized siRNA delivery system (denoted as M-MSN_siRNA@PEI-KALA), which is shown in Fig. 15B (a). These delivery vehicles possessed a notable siRNA protective effect and little cytotoxicity. They could be internalized easily into cells with an excellent endosomal escape capability, thereby successfully releasing the loaded siRNA into the cytoplasm to mediate remarkable interference effect on the target gene of the tumor cells. In the in vivo experiments, these vehicles significantly inhibited the tumor growth (Fig. 15B (b)), showing their potential application in cancer treatment.

MNPs and QDs, on the other hand, are excellent imaging reagents, and their coupling with drug delivery contributes to the combination of cancer diagnosis and therapy. There are several significant advantages for this combination:²⁰⁰ (1) imaging-guided chemotherapy helps monitor and quantify drug release. (2) The biodistribution and the target site accumulation of the therapeutic agents can be non-invasively assessed in real time. (3) Relying on the first two advantages, researchers can regulate and optimize the therapeutic interventions in a timely manner. (4) Imaging can also help to predict therapeutic responses, and evaluate the drug efficacy longitudinally. Therefore, imaging-guided drug delivery, facilitating the integration of diagnosis and treatment, can not only improve disease diagnosis and therapy, but also greatly promote the study of medical efficacy and toxicity, which would help us to better understand the process of drug delivery. Reddy et al.²⁰⁹ gave a schematic diagram (Fig. 16A) to show the general structure of the multifunctional device with magnetism, fluorescence, drug loading and bio-targeting, which can be used for simultaneous imaging, therapy, and cell sorting. Yu et al.²⁴ reported anti-biofouling polymer-coated, thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPIONs) loaded with doxorubicin (Dox; an anticancer drug) for combined cancer imaging and therapy in vivo (Fig. 16B and C). These Dox@TCL-SPIONs (Fig. 16B (a)) were intravenously injected into tumor-bearing mice, and then MR imaging was performed at scheduled time points to examine the localization and accumulation of the nanoparticles. At 4.5 h postinjection, noticeable darkening appeared in the tumor area, indicating a large accumulation of the nanoparticles within the tumor (Fig. 16B (b)), and in 24 h, most of the nanoparticles were removed from the tumor. The authors further compared the biodistribution of Dox in mice by fluorescence imaging for several major organs after intravenous injection of both Dox@TCL-SPIONs and free Dox (Fig. 16B (c and d)), which suggested that the Dox@TCL-SPIONs more readily accumulated in the tumor with much lower organ toxicity. Further in vivo experiments validated that the Dox@TCL-SPIONs showed remarkable inhibition of tumor growth of approximately 63%, which was even much higher than that of free Dox at an 8-fold higher dose (Fig. 16C (a)). Moreover, from the monitoring of mice body weight (Fig. 16C (b)) and the white blood cell number, a high dose of Dox exhibited severe toxicity, while the Dox@TCL-SPIONs was hardly toxic. Apart from combination with drugs, MNPs themselves are a kind of theranostic agent due to the fact that they can provide hyperthermia under an alternating magnetic field (AMF).^206,210,211 Compared with healthy cells, tumor cells are more sensitive to a temperature increase, and hence MNPs can be used to increase the temperature of tumor tissue in vivo and destroy pathological cells, which has fewer side effects than chemotherapy and radiotherapy. What's more, hyperthermia coupled with MRI can cause cancer treatment to proceed under monitoring in real time and in situ. In addition to direct tumor-cell killing, hyperthermia can also make tumor cells more susceptible to concomitant radio- or chemotherapy.^212–215 Thus combination of multiple therapies may achieve much better treatment efficacy. van der Zee et al.²¹⁶ compared radiotherapy alone and the radiotherapy plus hyperthermia in locally advanced pelvic tumors. The complete-response rates were 39% after radiotherapy and 55% after radiotherapy plus hyperthermia, and 3-year overall survival was 27% in the radiotherapy group and 51% in the radiotherapy plus hyperthermia group, which suggested that hyperthermia coupled with standard radiotherapy might be more useful in the treatment of locally advanced cervical tumors. Kim et al.,²¹⁷ Li et al.²¹⁸ and Quinto et al.²¹⁹ also found that the double effects of heat and drug were more effective than either chemotherapy or hyperthermia treatment alone. Now, combined therapy has become one of the most important development directions for cancer research, which has a great potential to improve the cure and survival rates of cancer patients, and in this field, nanocomposite-based drug delivery exhibits great application value, due to its easy manipulation, great versatility, good biocompatibility, and so on.

5. Conclusions and outlook

This review mainly outlined the preparation and the properties of the QD/MNP-based fluorescent/magnetic micro/nano-spheres, and further discussed their recent applications in cancer research in detail. Based on QDs and/or MNPs, fluorescent/magnetic micro/nano-spheres are constructed mainly through embedding the NPs into spheres, incorporating the NPs during the sphere formation process, assembling the NPs onto the surfaces of the spheres, or in situ synthesis of the NPs within the pores of the spheres. Each method has its own advantages and limitations, and researchers can select the appropriate one according to their demands and the available experimental conditions. The obtained NP–sphere composites not only inherit the unique fluorescence or magnetic properties of QDs or MNPs, but also show superiority over the NPs alone, for example, their high stability, strong signal, great versatility, good biocompatibility, convenient manipulation, and so on. Hence, the fluorescent/magnetic micro/nano-spheres exhibit great application value in cancer study: they can be used to isolate and enrich tumor cells or biomarkers from complex biofluids which greatly facilitates subsequent analyses; they can be utilized as imaging agents or reporter probes for cancer diagnosis; and they have now become an important platform for target drug delivery which may significantly improve the cure and survival rates of cancer patients.

Although much progress has been made with QD/MNP-based fluorescent/magnetic micro/nano-spheres, numerous challenges and issues remain to be resolved, and there is still a long way to go to substantiate their application in practice.^220–223 In terms of fabrication, novel synthetic methods still need to be explored to improve the problems with the current methods. To meet various application demands, NP–sphere composites with different morphology, different sizes (from nanometer to micrometer range), and different amounts of NPs have been pursued. Meanwhile, more convenient operation procedures and mass production are other important development directions. For the modification of spheres, orientation conjugation of functional molecules and precise control of their number are the main challenges. In addition, suitable surface coatings have been employed to improve the biocompatibility, specificity, and selectivity of the NP–sphere composites. As for their practical application, on the one hand, it's critical to develop a sound understanding of the thermodynamics and kinetics of the binding reaction at the sphere/solution interface, which will provide a theoretical foundation to guide their scientific application, such as better control of the formation of the sphere–biomolecule conjugates, easier optimizing of the operating conditions in targeted biological applications, and so on. On the other hand, to ensure the smooth transition from bench to the bedside, many issues must be addressed before the NP–sphere composites can be used in humans, including their biocompatibility, in vivo targeting efficacy, pharmacokinetics, biodistribution, toxicity, etc. Many researchers have been devoting themselves to these studies, and some have even made great progresses which, although are far from being ideal, show a very bright prospect for application.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 21535005, 21505157), the National Basic Research Program of China (863 Program, no. 2013AA032204), the 111 Project (111-2-10), Collaborative Innovation Center for Chemistry and Molecular Medicine, the Fund for Young Scientists of Shandong Province (no. BS2015SF001), and the Fundamental Research Funds for the Central Universities (no. 15CX02070A).

Notes and references

1. http://www.who.int/mediacentre/factsheets/fs297/en/ .
2. B. W. Stewart and C. P. Wild, IARC Nonserial Publication, 2014, ISBN 9789283204329.
3. X. Liu, Q. Dai, L. Austin, J. Coutts, G. Knowles, J. Zou, H. Chen and Q. Huo, J. Am. Chem. Soc., 2008, 130, 2780–2782 CrossRef CAS PubMed .
4. B. V. Chikkaveeraiah, A. A. Bhirde, N. Y. Morgan, H. S. Eden and X. Y. Chen, ACS Nano, 2012, 6, 6546–6561 CrossRef CAS PubMed .
5. X. Yu, H. S. Xia, Z. D. Sun, Y. Lin, K. Wang, J. Yu, H. Tang, D. W. Pang and Z. L. Zhang, Biosens. Bioelectron., 2013, 41, 129–136 CrossRef CAS PubMed .
6. T. M. Allen and P. R. Cullis, Science, 2004, 303, 1818–1822 CrossRef CAS PubMed .
7. R. Langer, Nature, 1998, 392, 5–10 CAS .
8. M. V. Yezhelyev, X. Gao, Y. Xing, A. Al-Hajj, S. M. Nie and R. M. O'Regan, Lancet Oncol., 2006, 7, 657–667 CrossRef CAS PubMed .
9. A. B. Chinen, C. M. Guan, J. R. Ferrer, S. N. Barnaby, T. J. Merkel and C. A. Mirkin, Chem. Rev., 2015, 115, 10530–10574 CrossRef CAS PubMed .
10. J. Yao, M. Yang and Y. Duan, Chem. Rev., 2014, 114, 6130–6178 CrossRef CAS PubMed .
11. D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit and R. Langer, Nat. Nanotechnol., 2007, 2, 751–760 CrossRef CAS PubMed .
12. X. Gao, Y. Cui, R. M. Levenson, L. W. Chung and S. Nie, Nat. Biotechnol., 2004, 22, 969–976 CrossRef CAS PubMed .
13. S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. V. Elst and R. N. Muller, Chem. Rev., 2008, 108, 2064–2110 CrossRef CAS PubMed .
14. L. Borlido, A. M. Azevedo, A. C. A. Roque and M. R. Aires-Barros, Biotechnol. Adv., 2013, 31, 1374–1385 CrossRef CAS PubMed .
15. I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi, Nat. Mater., 2005, 4, 435–446 CrossRef CAS PubMed .
16. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir and S. Weiss, Science, 2005, 307, 538–544 CrossRef CAS PubMed .
17. M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos, Science, 1998, 281, 2013–2016 CrossRef CAS .
18. A. H. Lu, E. L. Salabas and F. Schüth, Angew. Chem., Int. Ed., 2007, 46, 1222–1244 CrossRef CAS PubMed .
19. D. L. Leslie-Pelecky and R. D. Rieke, Chem. Mater., 1996, 8, 1770–1783 CrossRef CAS .
20. M. Colombo, S. Carregal-Romero, M. F. Casula, L. Gutiérrez, M. P. Morales, I. B. Böhm, J. T. Heverhagen, D. Prosperi and W. J. Parak, Chem. Soc. Rev., 2012, 41, 4306–4334 RSC .
21. X. Wu, H. Liu, J. Liu, K. N. Haley, J. A. Treadway, J. P. Larson, N. Ge, F. Peale and M. P. Bruchez, Nat. Biotechnol., 2002, 21, 41–46 CrossRef PubMed .
22. J. T. Cao, P. H. Zhang, Y. M. Liu, E. S. Abdel-Halim and J. J. Zhu, ACS Appl. Mater. Interfaces, 2015, 7, 14878–14884 CAS .
23. Y. W. Jun, Y. M. Huh, J. S. Choi, J. H. Lee, H. T. Song, S. Kim, S. Yoon, K. S. Kim, J. S. Shin, J. S. Suh and J. Cheon, J. Am. Chem. Soc., 2005, 127, 5732–5733 CrossRef CAS PubMed .
24. M. K. Yu, Y. Y. Jeong, J. Park, S. Park, J. W. Kim, J. J. Min, K. Kim and S. Jon, Angew. Chem., Int. Ed., 2008, 47, 5362–5365 CrossRef CAS PubMed .
25. P. Zrazhevskiy, M. Sena and X. Gao, Chem. Soc. Rev., 2010, 39, 4326–4354 RSC .
26. L. Qu and X. Peng, J. Am. Chem. Soc., 2002, 124, 2049–2055 CrossRef CAS PubMed .
27. L. Qu, Z. A. Peng and X. Peng, Nano Lett., 2001, 1, 333–337 CrossRef CAS .
28. Z. A. Peng and X. Peng, J. Am. Chem. Soc., 2001, 123, 183–184 CrossRef CAS PubMed .
29. A. M. Smith, S. Dave, S. M. Nie, L. True and X. H. Gao, Expert Rev. Mol. Diagn., 2006, 6, 231–244 CrossRef CAS PubMed .
30. T. Chen, I. Öçsoy, Q. Yuan, R. Wang, M. You, Z. Zhao, E. Song, X. Zhang and W. Tan, J. Am. Chem. Soc., 2012, 134, 13164–13167 CrossRef CAS PubMed .
31. J. Qian and X. Gao, ACS Appl. Mater. Interfaces, 2013, 5, 2845–2852 CAS .
32. J. Wang, S. Han, D. Ke and R. Wang, J. Nanomater., 2012, 2012, 1–8 Search PubMed .
33. M. Han, X. Gao, J. Z. Su and S. Nie, Nat. Biotechnol., 2001, 19, 631–635 CrossRef CAS PubMed .
34. H. Y. Xie, C. Zuo, Y. Liu, Z. L. Zhang, D. W. Pang, X. L. Li, J. P. Gong, C. Dickinson and W. Z. Zhou, Small, 2005, 1, 506–509 CrossRef CAS PubMed .
35. T. R. Sathe, A. Agrawal and S. Nie, Anal. Chem., 2006, 78, 5627–3562 CrossRef CAS PubMed .
36. N. Insin, J. B. Tracy, H. Lee, J. P. Zimmer, R. M. Westervelt and M. G. Bawendi, ACS Nano, 2008, 2, 197–202 CrossRef CAS PubMed .
37. H. L. Ding, Y. X. Zhang, S. Wang, J. M. Xu, S. C. Xu and G. H. Li, Chem. Mater., 2012, 24, 4572–4580 CrossRef CAS .
38. M. Xie, J. Hu, C. Y. Wen, Z. L. Zhang, H. Y. Xie and D. W. Pang, Nanotechnology, 2012, 23, 035602 CrossRef PubMed .
39. J. M. Shen, X. M. Guan, X. Y. Liu, J. F. Lan, T. Cheng and H. X. Zhang, Bioconjugate Chem., 2012, 23, 1010–1021 CrossRef CAS PubMed .
40. N. C. Bigall, W. J. Parak and D. Dorfs, Nano Today, 2012, 7, 282–296 CrossRef CAS .
41. A. R. K. Sasikala, A. GhavamiNejad, A. R. Unnithan, R. G. Thomas, M. Moon, Y.Y. Jeong, C. H. Park and C. S. Kim, Nanoscale, 2015, 7, 18119–18128 RSC .
42. Y. Liu, L. Liu, Y. He, L. Zhu and H. Ma, Anal. Chem., 2015, 87, 5286–5293 CrossRef CAS PubMed .
43. E. Song, W. Han, J. Li, Y. Jiang, D. Cheng, Y. Song, P. Zhang and W. Tan, Anal. Chem., 2014, 86, 9434–9442 CrossRef CAS PubMed .
44. K. Ming, J. Kim, M. J. Biondi, A. Syed, K. Chen, A. Lam, M. Ostrowski, A. Rebbapragada, J. J. Feld and W. C. W. Chan, ACS Nano, 2015, 9, 3060–3074 CrossRef CAS PubMed .
45. H. Y. Xie, M. Xie, Z. L. Zhang, Y. M. Long, X. Liu, M. L. Tang, D. W. Pang, Z. Tan, C. Dickinson and W. Z. Zhou, Bioconjugate Chem., 2007, 18, 1749–1755 CrossRef CAS PubMed .
46. G. P. Wang, E. Q. Song, H. Y. Xie, Z. L. Zhang, Z. Q. Tian, C. Zuo, D. W. Pang, D. C. Wu and Y. B. Shi, Chem. Commun., 2005, 4276–4278,  10.1039/b508075d .
47. E. Q. Song, G. P. Wang, H. Y. Xie, Z. L. Zhang, J. Hu, J. Peng, D. C. Wu, Y. B. Shi and D. W. Pang, Clin. Chem., 2007, 53, 2177–2185 CAS .
48. M. Xie, J. Hu, Y. M. Long, Z. L. Zhang, H. Y. Xie and D. W. Pang, Biosens. Bioelectron., 2009, 24, 1311–1317 CrossRef CAS PubMed .
49. J. Hu, M. Xie, C. Y. Wen, Z. L. Zhang, H. Y. Xie, A. A. Liu, Y. Y. Chen, S. M. Zhou and D. W. Pang, Biomaterials, 2011, 32, 1177–1184 CrossRef CAS PubMed .
50. E. Q. Song, J. Hu, C. Y. Wen, Z. Q. Tian, X. Yu, Z. L. Zhang, Y. B. Shi and D. W. Pang, ACS Nano, 2011, 5, 761–770 CrossRef CAS PubMed .
51. C. Y. Wen, J. Hu, Z. L. Zhang, Z. Q. Tian, G. P. Ou, Y. L. Liao, Y. Li, M. Xie, Z. Y. Sun and D. W. Pang, Anal. Chem., 2013, 85, 1223–1230 CrossRef CAS PubMed .
52. M. Wu, Z. L. Zhang, G. Chen, C. Y. Wen, L. L. Wu, J. Hu, C. C. Xiong, J. J. Chen and D. W. Pang, Small, 2015, 11, 5280–5288 CrossRef CAS PubMed .
53. C. Y. Wen, L. L. Wu, Z. L. Zhang, Y. L. Liu, S. Z. Wei, J. Hu, M. Tang, E. Z. Sun, Y. P. Gong, J. Yu and D. W. Pang, ACS Nano, 2014, 8, 941–949 CrossRef CAS PubMed .
54. J. Hu, C. Y. Wen, Z. L. Zhang, M. Xie, J. Hu, M. Wu and D. W. Pang, Anal. Chem., 2013, 85, 11929–11935 CrossRef CAS PubMed .
55. J. Hu, C. Y. Wen, Z. L. Zhang, M. Xie, H. Y. Xie and D. W. Pang, Biophys. J., 2014, 107, 165–173 CrossRef CAS PubMed .
56. J. J. Wang, Y. Z. Jiang, Y. Lin, L. Wen, C. Lv, Z. L. Zhang, G. Chen and D. W. Pang, Anal. Chem., 2015, 87, 11105–11112 CrossRef CAS PubMed .
57. R. Q. Zhang, S. L. Liu, W. Zhao, W. P. Zhang, X. Yu, Y. Li, A. J. Li, D. W. Pang and Z. L. Zhang, Anal. Chem., 2013, 85, 2645–2651 CrossRef CAS PubMed .
58. W. Zhao, W. P. Zhang, Z. L. Zhang, R. L. He, Y. Lin, M. Xie, H. Z. Wang and D. W. Pang, Anal. Chem., 2012, 84, 2358–2365 CrossRef CAS PubMed .
59. P. L. Guo, M. Tang, S. L. Hong, X. Yu, D. W. Pang and Z. L. Zhang, Biosens. Bioelectron., 2015, 74, 628–636 CrossRef CAS PubMed .
60. X. Yu, C. Y. Wen, Z. L. Zhang and D. W. Pang, RSC Adv., 2014, 4, 17660–17666 RSC .
61. N. Gaponik, I. L. Radtchenko, G. B. Sukhorukov, H. Weller and A. L. Rogach, Adv. Mater., 2002, 14, 879–882 CrossRef CAS .
62. N. Gaponik, I. L. Radtchenko, M. R. Gerstenberger, Y. A. Fedutik, G. B. Sukhorukov and A. L. Rogach, Nano Lett., 2003, 3, 369–372 CrossRef CAS .
63. D. W. Pang, H. Y. Xie, Y. Liu, C. Zuo and Z. L. Zhang, CN Patent200310111290.9, 2003 Search PubMed .
64. M. Bradley, N. Bruno and B. Vincent, Langmuir, 2005, 21, 2750–2753 CrossRef CAS PubMed .
65. S. V. Vaidya, A. Couzis and C. Maldarelli, Langmuir, 2015, 31, 3167–3179 CrossRef CAS PubMed .
66. P. O'Brien, S. S. Cummins, D. Darcy, A. Dearden, O. Masala, N. L. Pickett, S. Ryley and A. J. Sutherland, Chem. Commun., 2003, 2532–2533 RSC .
67. Y. Li, E. C. Y. Liu, N. Pickett, P. J. Skabara, S. S. Cummins, S. Ryley, A. J. Sutherland and P. O'Brien, J. Mater. Chem., 2005, 15, 1238–1243 CAS .
68. W. Sheng, S. Kim, J. Lee, S.-W. Kim, K. Jensen and M. G. Bawendi, Langmuir, 2006, 22, 3782–3790 CrossRef CAS PubMed .
69. K. Matsuyama, Y. Maeda, T. Matsuda, T. Okuyama and H. Muto, J. Supercrit. Fluids, 2015, 103, 83–89 CrossRef CAS .
70. N. Joumaa, M. Lansalot, A. Théretz, A. Elaissari, A. Sukhanova, M. Artemyev, I. Nabiev and J. H. M. Cohen, Langmuir, 2006, 22, 1810–1816 CrossRef CAS PubMed .
71. X. Yang and Y. Zhang, Langmuir, 2004, 20, 6071–6073 CrossRef CAS PubMed .
72. G. Wang, Y. Leng, H. Dou, L. Wang, W. Li, X. Wang, K. Sun, L. Shen, X. Yuan and J. Li, ACS Nano, 2013, 7, 471–481 CrossRef CAS PubMed .
73. Y. Li, Y. Wu, C. Luo, F. Yang, L. Qin, T. Fu, G. Wei, X. Kang and D. Wu, J. Mater. Chem. B, 2013, 1, 4644–4654 RSC .
74. Y. Chan, J. P. Zimmer, M. Stroh, J. S. Steckel, R. K. Jain and M. G. Bawendi, Adv. Mater., 2004, 16, 2092–2097 CrossRef CAS .
75. H. Zhang, Z. Cui, Y. Wang, K. Zhang, X. Ji, C. Lü, B. Yang and M. Gao, Adv. Mater., 2003, 15, 777–780 CrossRef CAS .
76. C. Zhou, M. Mao, H. Yuan, H. Shen, F. Wu, L. Ma and L. S. Li, J. Nanopart. Res., 2013, 15, 1901 CrossRef .
77. J. W. Kim, A. S. Utada, A. Fernández-Nieves, Z. Hu and D. A. Weitz, Angew. Chem., Int. Ed., 2007, 46, 1819–1822 CrossRef CAS PubMed .
78. Y. Zhao, H. C. Shum, H. Chen, L. L. A. Adams, Z. Gu and D. A. Weitz, J. Am. Chem. Soc., 2011, 133, 8790–8793 CrossRef CAS PubMed .
79. Y. Chen, P. F. Dong, J. H. Xu and G. S. Luo, Langmuir, 2014, 30, 8538–8542 CrossRef CAS PubMed .
80. X. Yu, G. Cheng, M. D. Zhou and S. Y. Zheng, Langmuir, 2015, 31, 3982–3992 CrossRef CAS PubMed .
81. Y. Zhao, Y. Cheng, L. Shang, J. Wang, Z. Xie and Z. Gu, Small, 2015, 11, 151–174 CrossRef CAS PubMed .
82. S. V. Vaidya, M. L. Gilchrist, C. Maldarelli and A. Couzis, Anal. Chem., 2007, 79, 8520–8530 CrossRef CAS PubMed .
83. Y. Q. Wang, Y. Y. Zhang, F. Zhang and W. Y. Li, J. Mater. Chem., 2011, 21, 6556–6562 RSC .
84. R. Wilson, D. G. Spiller, I. A. Prior, R. Bhatt and A. Hutchinson, J. Mater. Chem., 2007, 17, 4400–4406 RSC .
85. C. N. Allen, N. Lequeux, C. Chassenieux, G. Tessier and B. Dubertret, Adv. Mater., 2007, 19, 4420–4425 CrossRef CAS .
86. L. L. del Mercato, A. Z. Abbasi, M. Ochs and W. J. Parak, ACS Nano, 2011, 5, 9668–9674 CrossRef CAS PubMed .
87. S. Rauf, A. Glidle and J. M. Cooper, Adv. Mater., 2009, 21, 4020–4024 CrossRef CAS .
88. S. Rauf, A. Glidle and J. M. Cooper, Chem. Commun., 2010, 46, 2814–2816 RSC .
89. T. Hirai, T. Saito and I. Komasawa, J. Phys. Chem. B, 2001, 105, 9711–9714 CrossRef CAS .
90. R. Wilson, D. G. Spiller, I. A. Prior, K. J. Veltkamp and A. Hutchinson, ACS Nano, 2007, 1, 487–493 CrossRef CAS PubMed .
91. A. Berge, T. Ellingsen, O. B. Helgee and J. Ugelstad, US Patent4774265, 1988 Search PubMed .
92. A. Y. Gervald, I. A. Gritskova and N. I. Prokopov, Russ. Chem. Rev., 2010, 79, 219–229 CrossRef CAS .
93. http://www.thermofisher.com/cn/zh/home/brands/product-brand/dynal.html .
94. Q. Yang, Y. Li, T. Song and J. Chang, J. Mater. Chem., 2012, 22, 7043–7049 RSC .
95. A. Grützkau and A. Radbruch, Cytometry, Part A, 2010, 77A, 643–647 CrossRef PubMed .
96. C. Y. Nie, B. Wang, J. Y. Zhang, Y. Q. Cheng, F. T. Lv, L. B. Liu and S. Wang, Small, 2015, 11, 2555–2563 CrossRef CAS PubMed .
97. R. Hao, R. Xing, Z. Xu, Y. Hou, S. Gao and S. Sun, Adv. Mater., 2010, 22, 2729–2742 CrossRef CAS PubMed .
98. C. Kaewsaneha, P. Tangboriboonrat, D. Polpanich and A. Elaissari, ACS Appl. Mater. Interfaces, 2015, 7, 23373–23386 CAS .
99. Y. Shi, A. Pramanik, C. Tchounwou, F. Pedraza, R. A. Crouch, S. R. Chavva, A. Vangara, S. S. Sinha, S. Jones, D. Sardar, C. Hawker and P. C. Ray, ACS Appl. Mater. Interfaces, 2015, 7, 10935–10943 CAS .
100. L. Wang, W. Zhao, M. B. O'Donoghu and W. Tan, Bioconjugate Chem., 2007, 18, 297–301 CrossRef CAS PubMed .
101. A. J. Kell, G. Stewart, S. Ryan, R. Peytavi, M. Boissinot, A. Huletsky, M. G. Bergeron and B. Simard, ACS Nano, 2008, 2, 1777–1788 CrossRef CAS PubMed .
102. Y. Xing, Q. Chaudry, C. Shen, K. Y. Kong, H. E. Zhau, L. W. Chung, J. A. Petros, R. M. O'Regan, M. V. Yezhelyev, J. W. Simons, M. D. Wang and S. Nie, Nat. Protoc., 2007, 2, 1152–1165 CrossRef CAS PubMed .
103. E. Yilmaz, R. M. Alosmanov and M. Soylak, RSC Adv., 2015, 5, 33801–33808 RSC .
104. A. E. Chávez-Guajardo, J. C. Medina-Llamas, L. Maqueira, C. A. S. Andrade, K. G. B. Alves and C. P. de Melo, Chem. Eng. J., 2015, 281, 826–836 CrossRef .
105. L. Bai, Z. Li, Y. Zhang, T. Wang, R. Lu, W. Zhou, H. Gao and S. Zhang, Chem. Eng. J., 2015, 279, 757–766 CrossRef CAS .
106. L. Zhou, C. Gao and W. Xu, ACS Appl. Mater. Interfaces, 2010, 2, 1483–1491 CAS .
107. X. Li, W. Zhao, J. Gu, Y. Li, L. Li, D. Niu and J. Shi, Microporous Mesoporous Mater., 2015, 207, 142–148 CrossRef CAS .
108. Z. C. Xiong, Y. J. Chen, L. Y. Zhang, J. Ren, Q. Q. Zhang, M. L. Ye, W. B. Zhang and H. F. Zou, ACS Appl. Mater. Interfaces, 2014, 6, 22743–22750 CAS .
109. J. H. Gao, L. H. Li, P. L. Ho, G. C. Mak, H. W. Gu and B. Xu, Adv. Mater., 2006, 18, 3145–3148 CrossRef CAS .
110. S. M. Jo, J. j. Lee, W. Heu and H. S. Kim, Small, 2015, 11, 1975–1982 CrossRef CAS PubMed .
111. M. Xie, N. N. Lu, S. B. Cheng, X. Y. Wang, M. Wang, S. Guo, C. Y. Wen, J. Hu, D. W. Pang and W. H. Huang, Anal. Chem., 2014, 86, 4618–4626 CrossRef CAS PubMed .
112. H. Min, S. M. Jo and H. S. Kim, Small, 2015, 11, 2536–2542 CrossRef CAS PubMed .
113. J. H. Kang, S. Krause, H. Tobin, A. Mammoto, M. Kanapathipillai and D. E. Ingber, Lab Chip, 2012, 12, 2175–2181 RSC .
114. S. K. Arya, B. Lim and A. R. A. Rahman, Lab Chip, 2013, 13, 1995–2027 RSC .
115. M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. M. M. Terstappen and D. F. Hayes, N. Engl. J. Med., 2004, 351, 781–791 CrossRef CAS PubMed .
116. M. Cristofanilli, D. F. Hayes, G. T. Budd, M. J. Ellis, A. Stopeck, J. M. Reuben, G. V. Doyle, J. Matera, W. J. Allard, M. C. Miller, H. A. Fritsche, G. N. Hortobagyi and L. W. M. M. Terstappen, J. Clin. Oncol., 2005, 23, 1420–1430 CrossRef PubMed .
117. C. L. Chaffer and R. A. Weinberg, Science, 2011, 331, 1559–1564 CrossRef CAS PubMed .
118. P. Paterlini-Brechot and N. L. Benali, Cancer Lett., 2007, 253, 180–204 CrossRef CAS PubMed .
119. S. Nagrath, L. V. Sequist, S. Maheswaran, D. W. Bell, D. Irimia, L. Ulkus, M. R. Smith, E. L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U. J. Balis, R. G. Tompkins, D. A. Haber and M. Toner, Nature, 2007, 450, 1235–1239 CrossRef CAS PubMed .
120. M. Yu, S. Stott, M. Toner, S. Maheswaran and D. A. Haber, J. Cell Biol., 2011, 192, 373–382 CrossRef CAS PubMed .
121. R. Harouaka, Z. G. Kang, S. Y. Zheng and L. Cao, Pharmacol. Ther., 2014, 141, 209–221 CrossRef CAS PubMed .
122. S. Riethdorf, H. Fritsche, V. Muller, T. Rau, C. Schindlbeck, B. Rack, W. Janni, C. Coith, K. Beck, F. Janicke, S. Jackson, T. Gornet, M. Cristofanilli and K. Pantel, Clin. Cancer Res., 2007, 13, 920–928 CrossRef CAS PubMed .
123. E. Andreopoulou, L. Y. Yang, K. M. Rangel, J. M. Reuben, L. Hsu, S. Krishnamurthy, V. Valero, H. A. Fritsche and M. Cristofanilli, Int. J. Cancer, 2012, 130, 1590–1597 CrossRef CAS PubMed .
124. N. Beije, A. Jager and S. Sleijfer, Cancer Treat. Rev., 2015, 41, 144–150 CrossRef PubMed .
125. An Introduction to the CellSearch™ System.
126. N. N. Lu, M. Xie, J. Wang, S. W. Lv, J. S. Yi, W. G. Dong and W. H. Huang, ACS Appl. Mater. Interfaces, 2015, 7, 8817–8826 CAS .
127. K. Kerlikowske, A. M. Molinaro, M. L. Gauthier, H. K. Berman, F. Waldman, J. Bennington, H. Sanchez, C. Jimenez, K. Stewart, K. Chew, B. M. Ljung and T. D. Tlsty, J. Natl. Cancer Inst., 2010, 102, 627–637 CrossRef CAS PubMed .
128. J. A. Luna Coronell, P. Syed, K. Sergelen, I. Gyurján and A. Weinhäusel, J. Proteomics, 2012, 76, 102–115 CrossRef CAS PubMed .
129. J. L. Gong, Y. Liang, Y. Huang, J. W. Chen, J. H. Jiang, G. L. Shen and R. Q. Yu, Biosens. Bioelectron., 2007, 22, 1501–1507 CrossRef CAS PubMed .
130. S. Bi, Y. M. Yan, X. Y. Yang and S. S. Zhang, Chem. – Eur. J., 2009, 15, 4704–4709 CrossRef CAS PubMed .
131. Y. Li, Z. Wu and Z. Liu, Analyst, 2015, 140, 4083–4088 RSC .
132. D. Wang, N. Gan, H. Zhang, T. Li, L. Qiao, Y. Cao, X. Su and S. Jiang, Biosens. Bioelectron., 2015, 65, 78–82 CrossRef CAS PubMed .
133. X. Zhou, P. Cao, Y. Zhu, W. Lu, N. Gu and C. Mao, Nat. Mater., 2015, 14, 1058–1064 CrossRef CAS PubMed .
134. N. Pamme, Lab Chip, 2006, 6, 24–38 RSC .
135. M. A. M. Gijs, Microfluid. Nanofluid., 2004, 1, 22–40 CAS .
136. http://www.b2b.invitrogen.com/site/us/en/home/brands/Dynal/Dynabeads-Types-and-Uses.html .
137. http://www.caca.org.cn/system/2013/2001/2025/011126295.shtml .
138. N. Lee and T. Hyeon, Chem. Soc. Rev., 2012, 41, 2575–2589 RSC .
139. X. Liu, H. Jiang, Y. Fang, W. Zhao, N. Wang and G. Zang, Anal. Chem., 2015, 87, 9163–9169 CrossRef CAS PubMed .
140. G. Nie, C. Li, L. Zhang and L. Wang, J. Mater. Chem. B, 2014, 2, 8321–8328 RSC .
141. S. Bamrungsap, T. Chen, M. I. Shukoor, Z. Chen, K. Sefah, Y. Chen and W. H. Tan, ACS Nano, 2012, 6, 3974–3981 CrossRef CAS PubMed .
142. T. H. Shin, Y. Choi, S. Kim and J. Cheon, Chem. Soc. Rev., 2015, 44, 4501–4516 RSC .
143. E. Peng, F. Wang and J. M. Xue, J. Mater. Chem. B, 2015, 3, 2241–2276 RSC .
144. S. Sharifi, H. Seyednejad, S. Laurent, F. Atyabi, A. A. Saei and M. Mahmoudi, Contrast Media Mol. Imaging, 2015, 10, 329–355 CrossRef CAS PubMed .
145. D. E. Lee, H. Koo, I. C. Sun, J. H. Ryu, K. Kim and I. C. Kwon, Chem. Soc. Rev., 2012, 41, 2656–2672 RSC .
146. R. Tang, J. P. Xue, B. G. Xu, D. W. Shen, G. P. Sudlow and S. Achilefu, ACS Nano, 2015, 9, 220–230 CrossRef CAS PubMed .
147. V. Pansare, S. Hejazi, W. Faenza and R. K. Prud'homme, Chem. Mater., 2012, 24, 812–827 CrossRef CAS PubMed .
148. Y. P. Gu, R. Cui, Z. L. Zhang, Z. X. Xie and D. W. Pang, J. Am. Chem. Soc., 2012, 134, 79–82 CrossRef CAS PubMed .
149. P. Jiang, C. N. Zhu, Z. L. Zhang, Z. Q. Tian and D. W. Pang, Biomaterials, 2012, 33, 5130–5135 CrossRef CAS PubMed .
150. C. N. Zhu, P. Jiang, Z. L. Zhang, D. L. Zhu, Z. Q. Tian and D. W. Pang, ACS Appl. Mater. Interfaces, 2013, 5, 1186–1189 CAS .
151. J. Xie, K. Chen, H. Y. Lee, C. Xu, A. R. Hsu, S. Peng, X. Chen and S. Sun, J. Am. Chem. Soc., 2008, 130, 7542–7543 CrossRef CAS PubMed .
152. L. Jing, K. Ding, S. V. Kershaw, I. M. Kempson, A. L. Rogach and M. Gao, Adv. Mater., 2014, 26, 6367–6386 CrossRef CAS PubMed .
153. J. Kim, Y. Piao and T. Hyeon, Chem. Soc. Rev., 2009, 38, 372–390 RSC .
154. S. Puttick, C. Bell, N. Dowson, S. Rose and M. Fay, Drug Discovery Today, 2015, 20, 306–317 CrossRef PubMed .
155. G. Zhang, R. Du, L. Zhang, D. Cai, X. Sun, Y. Zhou, J. Zhou, J. Qian, K. Zhong, K. Zheng, D. Kaigler, W. Liu, X. Zhang, D. Zou and Z. Wu, Adv. Funct. Mater., 2015, 25, 6101–6111 CrossRef CAS .
156. D. Le Sage, K. Arai, D. R. Glenn, S. J. DeVience, L. M. Pham, L. Rahn-Lee, M. D. Lukin, A. Yacoby, A. Komeili and R. L. Walsworth, Nature, 2013, 496, 486–491 CrossRef CAS PubMed .
157. J. Lin, Y. Li, Y. Li, H. Wu, F. Yu, S. Zhou, L. Xie, F. Luo, C. Lin and Z. Hou, ACS Appl. Mater. Interfaces, 2015, 7, 11908–11920 CAS .
158. J. Garcia, T. Tang and A. Y. Louie, Nanomedicine, 2015, 10, 1343–1359 CrossRef CAS PubMed .
159. X. Wang, D. Niu, P. Li, Q. Wu, X. Bo, B. Liu, S. Bao, T. Su, H. Xu and Q. Wang, ACS Nano, 2015, 9, 5646–5656 CrossRef CAS PubMed .
160. J. Huang, M. Guo, H. T. Ke, C. Zong, B. Ren, G. Liu, H. Shen, Y. F. Ma, X. Y. Wang, H. L. Zhang, Z. W. Deng, H. B. Chen and Z. J. Zhang, Adv. Mater., 2015, 27, 5049–5056 CrossRef CAS PubMed .
161. L. Y. Bai, X. Q. Yang, J. An, L. Zhang, K. Zhao, M. Y. Qin, B. Y. Fang, C. Li, Y. Xuan, X. S. Zhang, Y. D. Zhao and Z. Y. Ma, Nanotechnology, 2015, 26, 315701 CrossRef PubMed .
162. J. H. Park, G. v. Maltzahn, E. Ruoslahti, S. N. Bhatia and M. J. Sailor, Angew. Chem., 2008, 120, 7394–7398 CrossRef .
163. E. S. Shibu, K. Ono, S. Sugino, A. Nishioka, A. Yasuda, Y. Shigeri, S.-i. Wakida, M. Sawada and V. Biju, ACS Nano, 2013, 7, 9851–9859 CrossRef CAS PubMed .
164. A. Xia, Y. Gao, J. Zhou, C. Li, T. Yang, D. Wu, L. Wu and F. Li, Biomaterials, 2011, 32, 7200–7208 CrossRef CAS PubMed .
165. Y. Jiao, Y. Sun, X. Tang, Q. Ren and W. Yang, Small, 2015, 11, 1962–1974 CrossRef CAS PubMed .
166. C. Wang, Y. Yao and Q. Song, J. Mater. Chem. C, 2015, 3, 5910–5917 RSC .
167. H. Kobayashi, Y. Hama, Y. Koyama, T. Barrett, C. A. S. Regino, Y. Urano and P. L. Choyke, Nano Lett., 2007, 7, 1711–1716 CrossRef CAS PubMed .
168. Y. W. Jun, J. H. Lee and J. Cheon, Angew. Chem., Int. Ed., 2008, 47, 5122–5235 CrossRef CAS PubMed .
169. B. Gleich and J. Weizenecker, Nature, 2005, 435, 1214–1217 CrossRef CAS PubMed .
170. J. Weizenecker, B. Gleich, J. Rahmer, H. Dahnke and J. Borgert, Phys. Med. Biol., 2009, 54, L1–L10 CrossRef CAS PubMed .
171. J. Weizenecker, J. Borgert and B. Gleich, Phys. Med. Biol., 2007, 52, 6363–6374 CrossRef CAS PubMed .
172. S. Biederer, T. Knopp, T. F. Sattel, K. Lüdtke-Buzug, B. Gleich, J. Weizenecker, J. Borgert and T. M. Buzug, J. Phys. D: Appl. Phys., 2009, 42, 205007 CrossRef .
173. Z. Zhou, D. Huang, J. Bao, Q. Chen, G. Liu, Z. Chen, X. Chen and J. Gao, Adv. Mater., 2012, 24, 6223–6228 CrossRef CAS PubMed .
174. T.-H. Shin, J.-s. Choi, S. Yun, I.-S. Kim, H.-T. Song, Y. Kim, K. I. Park and J. Cheon, ACS Nano, 2014, 8, 3393–3401 CrossRef CAS PubMed .
175. H. Lee, M. K. Yu, S. Park, S. Moon, J. J. Min, Y. Y. Jeong, H.-W. Kang and S. Jon, J. Am. Chem. Soc., 2007, 129, 12739–12745 CrossRef CAS PubMed .
176. O. Veiseh, C. Sun, C. Fang, N. Bhattarai, J. Gunn, F. Kievit, K. Du, B. Pullar, D. Lee, R. G. Ellenbogen, J. Olson and M. Zhang, Cancer Res., 2009, 69, 6200–6207 CrossRef CAS PubMed .
177. H. Y. Lee, Z. Li, K. Chen, A. R. Hsu, C. Xu, J. Xie, S. Sun and X. Chen, J. Nucl. Med., 2008, 49, 1371–1379 CrossRef CAS PubMed .
178. L. Sandiford, A. Phinikaridou, A. Protti, L. K. Meszaros, X. Cui, Y. Yan, G. Frodsham, P. A. Williamson, N. Gaddum, R. M. Botnar, P. J. Blower, M. A. Green and R. T. M. de Rosales, ACS Nano, 2013, 7, 500–512 CrossRef CAS PubMed .
179. R. Torres Martin de Rosales, R. Tavare, R. L. Paul, M. Jauregui-Osoro, A. Protti, A. Glaria, G. Varma, I. Szanda and P. J. Blower, Angew. Chem., Int. Ed., 2011, 50, 5509–5513 CrossRef PubMed .
180. R. Madru, P. Kjellman, F. Olsson, K. Wingardh, C. Ingvar, F. Stahlberg, J. Olsrud, J. Latt, S. Fredriksson, L. Knutsson and S. E. Strand, J. Nucl. Med., 2012, 53, 459–463 CrossRef CAS PubMed .
181. J. T. Wang, L. Cabana, M. Bourgognon, H. Kafa, A. Protti, K. Venner, A. M. Shah, J. Sosabowski, S. J. Mather, A. Roig, X. Ke, G. V. Tendeloo, R. T. de Rosales, G. Tobias and K. T. Al-Jamal, Adv. Funct. Mater., 2014, 24, 1880–1894 CrossRef CAS PubMed .
182. Y. Sun, Y. Zheng, H. Ran, Y. Zhou, H. Shen, Y. Chen, H. Chen, T. M. Krupka, A. Li, P. Li, Z. Wang and Z. Wang, Biomaterials, 2012, 33, 5854–5864 CrossRef CAS PubMed .
183. C. Niu, Z. Wang, G. Lu, T. M. Krupka, Y. Sun, Y. You, W. Song, H. Ran, P. Li and Y. Zheng, Biomaterials, 2013, 34, 2307–2317 CrossRef CAS PubMed .
184. J. I. Park, D. Jagadeesan, R. Williams, W. Oakden, S. Chung, G. J. Stanisz and E. Kumacheva, ACS Nano, 2010, 4, 6579–6586 CrossRef CAS PubMed .
185. Y. Fang, L. Ling, L. Yixin, C. Zhongping, W. Junru and G. Ning, Phys. Med. Biol., 2008, 53, 6129–6141 CrossRef PubMed .
186. H. W. Yang, H. L. Liu, M. L. Li, I. W. Hsi, C. T. Fan, C. Y. Huang, Y. J. Lu, M. Y. Hua, H. Y. Chou, J. W. Liaw, C. C. Ma and K. C. Wei, Biomaterials, 2013, 34, 5651–5660 CrossRef CAS PubMed .
187. J. Yu, C. Yang, J. Li, Y. Ding, L. Zhang, M. Z. Yousaf, J. Lin, R. Pang, L. Wei, L. Xu, F. Sheng, C. Li, G. Li, L. Zhao and Y. Hou, Adv. Mater., 2014, 26, 4114–4120 CrossRef CAS PubMed .
188. X. R. Song, X. Wang, S. X. Yu, J. Cao, S. H. Li, J. Li, G. Liu, H. H. Yang and X. Chen, Adv. Mater., 2015, 27, 3285–3291 CrossRef CAS PubMed .
189. X. J. Zhao, L. R. Hilliard, S. J. Mechery, Y. P. Wang, R. P. Bagwe, S. G. Jin and W. H. Tan, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 15027–15032 CrossRef CAS PubMed .
190. Y. Leng, K. Sun, X. Chen and W. Li, Chem. Soc. Rev., 2015, 44, 5552–5595 RSC .
191. S. W. Han, E. Jang and W. G. Koh, Sens. Actuators, B, 2015, 209, 242–251 CrossRef CAS .
192. K. Brazhnik, Z. Sokolova, M. Baryshnikova, R. Bilan, A. Efimov, I. Nabiev and A. Sukhanova, Nanomed.: Nanotechnol., Biol. Med., 2015, 11, 1065–1075 CrossRef CAS PubMed .
193. A. Sukhanova and I. Nabiev, Crit. Rev. Oncol. Hematol., 2008, 68, 39–59 CrossRef PubMed .
194. O. Akinfieva, I. Nabiev and A. Sukhanova, Crit. Rev. Oncol. Hematol., 2013, 86, 1–14 CrossRef PubMed .
195. A. Z. Abbasi, F. Amin, T. Niebling, S. Friede, M. Ochs, S. Carregal-Romero, J.-M. Montenegro, P. Rivera Gil, W. Heimbrodt and W. J. Parak, ACS Nano, 2011, 5, 21–25 CrossRef CAS PubMed .
196. U. Kaiser, D. Jimenez de Aberasturi, R. Malinowski, F. Amin, W. J. Parak and W. Heimbrodt, Appl. Phys. Lett., 2014, 104, 041901 CrossRef .
197. B. Srinivasan, Y. P. Li, Y. Jing, Y. H. Xu, X. F. Yao, C. G. Xing and J. P. Wang, Angew. Chem., Int. Ed., 2009, 48, 2764–2767 CrossRef CAS PubMed .
198. A. Manteca, M. Mujika and S. Arana, Biosens. Bioelectron., 2011, 26, 3705–3709 CrossRef CAS PubMed .
199. S. Q. Tang, S. J. Moon, K. H. Park, S. H. Paek, K. W. Chung and S. Bae, J. Nanosci. Nanotechnol., 2011, 11, 82–89 CrossRef CAS PubMed .
200. T. Lammers, F. Kiessling, W. E. Hennink and G. Storm, Mol. Pharmaceutics, 2010, 7, 1899–1912 CrossRef CAS PubMed .
201. Y. Omidi, BioImpacts, 2011, 1, 145–147 CAS .
202. G. Chen, J. Y. Zhu, Z. L. Zhang, W. Zhang, J. G. Ren, M. Wu, Z. Y. Hong, C. Lv, D. W. Pang and Y. F. Zhao, Angew. Chem., Int. Ed., 2015, 54, 1036–1040 CrossRef CAS PubMed .
203. Y. Wang and H. Gu, Adv. Mater., 2015, 27, 576–585 CrossRef CAS PubMed .
204. S. K. Yen, P. Padmanabhan and S. T. Selvan, Theranostics, 2013, 3, 986–1003 CrossRef CAS PubMed .
205. B. Cai, M. Zhao, Y. Ma, Z. Ye and J. Huang, ACS Appl. Mater. Interfaces, 2015, 7, 1327–1333 CAS .
206. C. S. Kumar and F. Mohammad, Adv. Drug Delivery Rev., 2011, 63, 789–808 CrossRef CAS PubMed .
207. C. R. Thomas, D. P. Ferris, J. H. Lee, E. Choi, M. H. Cho, E. S. Kim, J. F. Stoddart, J. S. Shin, J. Cheon and J. I. Zink, J. Am. Chem. Soc., 2010, 132, 10623–10625 CrossRef CAS PubMed .
208. X. Li, Y. Chen, M. Wang, Y. Ma, W. Xia and H. Gu, Biomaterials, 2013, 34, 1391–1401 CrossRef CAS PubMed .
209. L. H. Reddy, J. L. Arias, J. Nicolas and P. Couvreur, Chem. Rev., 2012, 112, 5818–5878 CrossRef CAS PubMed .
210. J. P. Fortin, C. Wilhelm, J. Servais, C. Ménager, J. C. Bacri and F. Gazeau, J. Am. Chem. Soc., 2007, 129, 2628–2635 CrossRef CAS PubMed .
211. S. Laurent, S. Dutz, U. O. Häfeli and M. Mahmoudi, Adv. Colloid Interface Sci., 2011, 166, 8–23 CrossRef CAS PubMed .
212. S. Kossatz, J. Grandke, P. Couleaud, A. Latorre, A. Aires, K. Crosbie-Staunton, R. Ludwig, H. Dähring, V. Ettelt, A. Lazaro-Carrillo, M. Calero, M. Sader, J. Courty, Y. Volkov, A. Prina-Mello, A. Villanueva, Á. Somoza, A. L. Cortajarena, R. Miranda and I. Hilger, Breast Cancer Res., 2015, 17, 66 CrossRef PubMed .
213. B. Thiesen and A. Jordan, Int. J. Hyperthermia, 2008, 24, 467–474 CrossRef CAS PubMed .
214. A. Ito, Y. Kuga, H. Honda, H. Kikkawa, A. Horiuchi, Y. Watanabe and T. Kobayashi, Cancer Lett., 2004, 212, 167–175 CrossRef CAS PubMed .
215. P. T. Yin, B. P. Shah and K. B. Lee, Small, 2014, 10, 4106–4112 CAS .
216. J. van der Zee, D. González, G. C. van Rhoon, J. D. P. van Dijk, W. L. J. van Putten and A. A. M. Hart, Lancet, 2000, 355, 1119–1125 CrossRef CAS .
217. H. C. Kim, E. Kim, S. W. Jeong, T. L. Ha, S. I. Park, S. G. Lee, S. J. Lee and S. W. Lee, Nanoscale, 2015, 7, 16470–16480 RSC .
218. J. H. Li, Y. Hu, Y. H. Hou, X. K. Shen, G. Q. Xu, L. L. Dai, J. Zhou, Y. Liu and K. Y. Cai, Nanoscale, 2015, 7, 9004–9012 RSC .
219. C. A. Quinto, P. Mohindra, S. Tong and G. Bao, Nanoscale, 2015, 7, 12728–12736 RSC .
220. J. G. Corbin and T. F. Haydar, Nanomedicine, 2007, 2, 579–581 CrossRef CAS PubMed .
221. K. Mandel and F. Hutter, Nano Today, 2012, 7, 485–487 CrossRef CAS .
222. J. M. Klostranec and W. C. W. Chan, Adv. Mater., 2006, 18, 1953–1964 CrossRef CAS .
223. E. K. Lim, T. Kim, S. Paik, S. Haam, Y. M. Huh and K. Lee, Chem. Rev., 2015, 115, 327–394 CrossRef CAS PubMed .

This journal is © The Royal Society of Chemistry 2016