Micro-flow assisted synthesis of fluorescent polymer nanoparticles with tuned size and surface properties

2.1 Process concept and components of the reactions system

When a synthesis of the materials is performed in a conventional batch reactor, all reactants are mixed together in a single vessel. Hence, all partial processes take place simultaneously in one reactor. On the other side, advantageous over conventional batch process, a microcontinuous flow process is the subdivision of a complex chemical process into the separate several steps. Fast mixing and efficient transport of the reactants makes the microfluidic technique a very efficient strategy for fast and controlled nucleation and growth in the synthesis of colloidal nanoparticles and support the formation of nanoparticles with narrow size distribution and in a high yield. Following individual steps are realized in the microreactor system for particles synthesis: (a) Individual flow of the aqueous and monomer phase interacts in cross-flow arrangement of microreactor. (b) Emulsion forms due to the presence of surfactant in aqueous phase. (c) Thermal source initiates the polymerization reaction in a flow-through heating block. (d) Nucleation and growth of the particles obtained via homogeneous nucleation mechanism. (e) Homogeneous PMMA nanoparticles obtained at the end of polymerization process. (f) Further functionalization of the polymer nanoparticles with different fluorophores makes the particles fluorescence active.

2.2 Microfluidic-assisted synthesis of PMMA nanoparticles

For the preparation of aqueous phase, 18 mg of cationic surfactant cetyltrimethylammonium bromide (CTAB) was dissolved in 50 ml of deionized water (1 mm). For different experiments, the concentration of CTAB has been varied from 1 mm down to 0.1 μm. The aqueous solution of CTAB has been filled up in a glass syringe and fixed to the syringe pump for actuation. In another glass syringe, 10 μl (1%) of ethylene glycol dimethacrylate (EGDMA) cross-linker and 3.5 mg thermal initiator azobisisobutyronitrile (AIBN) were dissolved in 990 μl of methyl methacrylate (MMA) as organic phase. Formation of the emulsion from two opposite phases started at the cross-point of the microreactor (shown in Figure 1) where monomer mixture meets a streaming aqueous phase of surfactant. An aqueous phase is guided through a capillary slit in perpendicular direction to the monomer flow. Small droplets of the monomer liquid are generated through dripping mode by the strong shear force of continuous phase. The emulsion solution was collected in a heated tube for completion of the polymerization reaction. The temperature of the heating block for polymerization reaction was maintained at 97°C. Size-controlled PMMA nanoparticles were obtained at the end of the polymerization reaction.

Figure 1:

(A) Schematic of the microfluidic setup and (B) scheme for the formation and growth of homogeneous polymer nanoparticles via emulsion polymerization.

2.3 Noncovalent binding of fluorophores in the nanoparticle interior

For the preparation of monomer phase, 0.1 mg Nile red (fluorescence dye) was dissolved in 1 ml of monomer mixture (MMA, EGDMA, and AIBN). Similar to the above procedure of nanoparticles synthesis, red-colored nanoparticles were obtained after completion of the polymerization reaction. Five different concentrations of SDS and CTAB surfactants (0.1 μm, 1 μm, 10 μm, 0.1 mm, and 1 mm) in the aqueous phase were used for tuning the particles size between 70 nm and 500 nm. Similarly, yellow-colored PMMA nanoparticles have been obtained when 12-(7-nitrobenzofuran-4-ylamino)dodecanoic acid (NBFD) dye was used in the monomer mixture.

2.4 Covalent-linking of fluorophores in the polymer nanoparticles

In another manner, a suitable fluorophore was covalently linked to the MMA molecules prior to the polymerization reaction. The synthesis of heterocyclic fluorophores bearing a MMA sub-moiety was reported earlier by our coworkers [23] and is described below briefly.

2.5 Fluorophore-functionalized PMMA nanoparticles

PMMA nanoparticles can become fluorescence active via two ways. In the first case, the aqueous phase is made up of the different concentrations of Titan yellow solution. Titan yellow, 34.5 mg, was dissolved in 50 ml of deionized water (1 mm) and has been filled up in a glass syringe, which served as the continuous phase. A similar reaction was performed where monomer droplets were generated via dripping mode, and nanoparticles were obtained at 97°C. Different concentrations of Titan yellow (1 μm to 1 mm) were used for tuning the nanoparticle diameter between 80 nm and 200 nm. In this single-step process, nanoparticles can be obtained with tuning in the size, surface charge, and fluorescence intensities simultaneously.

In the second case, layer-by-layer additional modification steps were applied on the CTAB-covered PMMA nanoparticles with polypeptides. At first, 1 mg of poly-l-glutamic acid (PGA) was dissolved in 1 ml of PBS buffer solution (pH 7.4). Then, centrifuged PMMA nanoparticles were dispersed in the PBS buffer solution of PGA with 1 h incubation time at moderate stirring rate. Afterward, the nanoparticles were washed and re-dispersed several times in de-ionized water. In the next step, 1 mg of poly-l-lysine (PLL) was dissolved in 1 ml of PBS buffer solution (pH 7.4) in a separate vial. PGA-covered PMMA nanoparticles were re-dispersed in PLL solution, and again, moderate stirring was maintained during the incubation for 1 h. After repeated washing and centrifugation process, particles were re-dispersed in 1 ml NHS-biotin solution, which is also prepared in PBS buffer solution. In the last step, a PBS buffer solution of fluorescence dye-labeled streptavidin was added to the PLL-covered PMMA nanoparticles. Blue-colored PMMA nanoparticles were obtained at the end via immobilization of fluorophore.

3.1 Size-tuned PMMA nanoparticles

Microfluidics is an emerging platform for the polymer particle synthesis at the nano- and microscale levels [19, 24]. In the droplet-based microfluidic system [25], the nucleation and growth process take place under very homogeneous conditions due to the advantages of this process such as fast phase transfer as well as efficient mixing of all reactants within a short time interval [18, 20]. Resultantly, the particulate product of polymeric materials was obtained with high homogeneity. By differently designed microreactors, the polymer particles with various functionalities have been reported in the literature [26–30]. Moreover, the polymer is an amorphous material, and therefore, to control the shape is a challenging task. The microreactor is even more interesting because it allows the possibility to control the shape of polymer particles in a single step [31–35]. In our work, the size-controlled spherical poly(methyl methacrylate) (PMMA) nanoparticles have been prepared in the cross-flow microfluidic setup as shown in Figure 1. A surfactant reduces the surface tension at the interface, and therefore, the size of the droplets (particles) can be controlled by different concentrations [36]. The strong effect of cationic CTAB concentration (between 0.1 μm and 1 mm) on the particle sizes is well illustrated in Figure 2. Smaller PMMA nanoparticles of about 70 nm were obtained at a higher CTAB concentration (1 mm), whereas larger nanoparticles of about 350 nm were generated at a lower CTAB concentration (0.1 μm). In this way, by keeping the constant flow rate ratio of both immiscible liquids (1200 μl/min/70 μl/min, aqueous/monomer), the size of the PMMA nanoparticles can be systematically tuned between about 70 nm and 350 nm at different CTAB concentrations (Figure 2A–E). Along with the size, the surface potential of the nanoparticles depends on the CTAB concentrations, too. A higher ζ potential of the nanoparticles (about +42 mV) was realized when 1 mm of CTAB was used in the aqueous phase. The surface charge of the particles gradually decreases with a decrease in CTAB concentration (Figure 2G). Similarly, simultaneous effects on the particle size and surface potential are obtained when cationic CTAB is replaced by anionic SDS. When 1 mm of SDS was used in the aqueous phase, the size of the obtained nanoparticles was about 105 nm (Figure 2F). The size of the particles increased up to about 500 nm when the SDS concentration gradually decreased down to 0.1 μm. The ζ potential of the nanoparticles can be tuned between about -5 mV and -24 mV with a variation in concentration of anionic SDS between 0.1 μm and 1 mm in the aqueous phase (Figure 2G).

Figure 2:

(A–E) SEM images of the size-tuned PMMA nanoparticles obtained when different concentrations of CTAB were used in the aqueous phase; (A) 1 mm, (B) 0.1 mm, (C) 0.01 mm, (D) 1 μm, and (E) 0.1 μm, (flow rate ratio of both immiscible liquids was 1200 μl/min/70 μl/min, aqueous/monomer). Scale bar for all five images are the same (1 μm). (F) and (G) are the graphical results for the nanoparticle size and surface charge (ζ potential) in dependence of the cationic CTAB and anionic SDS surfactants concentrations in the aqueous phase.

Initially, when an emulsion comes into contact with a heating temperature (97°C), the initiation for the nanoparticle nucleation takes place. Nucleation of the polymer particles can possibly occur via two different routes, a micelle nucleation or homogeneous nucleation. In the continuous phase, when the concentration of the surfactant is above the critical micelle concentration (CMC), the obtained particles follow the micelle nucleation mechanism. Otherwise, the formation of the particles is assisted by a homogeneous nucleation mechanism in the case when the concentration of the surfactant is below CMC. For example, the CMC of SDS is ~8 mm at 25°C [37]. Here, the concentrations of both surfactants (SDS and CTAB) are below CMC. Hence, the particles are obtained via the homogeneous nucleation mechanism route. Owing to the thermal energy at elevated temperature, initiation starts when a radical attacks on the monomers to form the nucleated particles. During the homogeneous nucleation, the adsorbed surfactant molecules may desorb out, and monomer addition (diffusion) takes place in the small nucleated particles to obtain primary growth [37]. When the polymerization is completed, the surfactant molecules are adsorbed irreversibly on the surface of final nanoparticles. The density of the surface charge, and therefore the size, depends on the surfactant concentration used in the aqueous phase (Figure 2F and G).

3.2 Fluorophores in the polymer interior

Fluorescence-based labels are a suitable technique for the luminescence and fluorescence imaging and sensing [11]. Types, sustainability, and surface properties of the labels should be selective for the application in the in vitro assays and in vivo imaging. Organic dyes can be a good choice for labeling purposes, but the non-optimum spectroscopic features and photochemical instability are the issues, which hinder them to be used in sensitive measurements [7]. However, when organic dyes and various fluorophores are embedded in the host matrix, they can be used for sensitive analysis owing to the increment of the fluorescence lifetime and brightness. Various methods, therefore, have been employed to incorporate the various fluorophores inside the polymer matrix such as, incorporation of fluorophore by swelling methods, ex situ binding over the surface via different interactions, and so forth [38, 39]. However, these methods are multistep processes and lack precise distribution of fluorophore inside the polymer matrixes. In our work, the fluorescent polymer nanoparticles were produced in a single-step process and with high fluorophore loading (Scheme 1). Fluorescence Nile red dye embeds in the polymer nanoparticles in a noncovalent dispersion way through the “like dissolves like” principle. The applications such as labeling, sensing, and materials for photonic crystals demand for the hydrophilic dispersion. In the obtained nanoparticles here, the surfactant (CTAB) layer on the surface of the particles allows them to become well dispersed in the aqueous phase. Nile red and 12-(7-nitrobenzofuran-4-ylamino)dodecanoic acid (NBFD)-embedded PMMA nanoparticles show fluorescence emission at 593 nm and 532 nm, respectively, as shown in Figure 3.

Scheme 1:

Schematic overview of the different strategies for preparing four different classes of fluorescent polymer nanoparticles via semi-microfluidic platform.

Figure 3:

(A) Camera picture of the Nile red (red) and NBFD (yellow) embedded colored PMMA nanoparticles suspension (smaller nanoparticles (extreme left bottle) are stable in dispersion and bigger particles (extreme right bottle) settled down at the bottom after 2 h), (B) the chemical structure of the used dyes, and (C) and (D) are the fluorescence spectra of the Nile red and NBFD-embedded PMMA nanoparticles, respectively.

On the other side, there is also growing interest for utilizing the fluorophores in organic medium. The dye doped polymer nanoparticles swell in the organic phase, and dye molecules, therefore, may leak in the surrounding. Because of such leaking, the brightness of the label particles decreased, and they photobleached at a longer time. Hence, the fluorescence lifetime and fluorescence yield of the nanoparticles decreased down drastically. To avoid these concerns of hydrophobic fluorescent polymer nanoparticles in the organic phase, the fluorophore should be covalently linked in a polymer network. The method, therefore, we have used here is by functionalizing the monomer with fluorophore via covalent linking prior to the polymerization process. An organic molecule 4-hydroxythiazol substructure was prepared as described earlier [23] by a Hantzsch-like reaction, employing 2-cyanopyridine and 2-mercaptopropionic acid under solvent-free conditions at elevated temperatures using pyridine as a catalyst. The functionalization of MMA with the heterocyclic molecule 5-methyl-2-(pyridin-2-yl)thiazol-4-ol forms 5-methyl-2-(pyridin-2-yl)thiazol-4-yl methacrylate (Scheme 2). Here, a microflow process has been performed to obtain the covalently linked fluorophore-PMMA nanoparticles of different sizes between 70 nm and 400 nm (Figure 4) where a different mixing ratio of fluorophore-functionalized MMA (heterocyclic acrylate molecule) and pure MMA has been used in the monomer phase. By varying the concentrations (weight ratio) of the fluorophore-functionalized MMA in the pure MMA solution and also by varying the SDS concentration in the aqueous phase at different flow rate ratios, the size and surface potential-tuned fluorescent polymer nanoparticles were obtained (Figure 4). Along with the size-controlled parameter, the shape-controlled and composite fluorescent polymer nanoparticles also have different applications, which has been explained in the below section.

Scheme 2:

Scheme represent the chemical synthesis of fluorophore-functionalized monomer 5-methyl-2-(pyridine-2-yl)thiazol-4-yl methacrylate molecule and polymerization reaction for the covalent linking of fluorophore in the nanoparticles interiors.

Figure 4:

(A–C) SEM images of the covalently linked fluorophore-embedded PMMA nanoparticles obtained when different concentrations of SDS, (A) 1 mm, (B) 0.1 mm, and (C) 0.01 mm in the aqueous phase were used (flow rate ratio 1200/70 μl/min, aqueous/monomer). Scale bar for all three SEM images are the same (500 nm). (d) Graphical representation of particles size dependent on SDS concentrations in the aqueous phase. (E) Fluorescence spectra of the obtained particles at different ratios of pure MMA and fluorophore-functionalized MMA in the monomer phase (controlled mixing of pure MMA and fluorophore functionalized MMA have been prepared in the monomer phase, and fluorescence spectra were measured after particle formation).

3.3 Nonspherical and composite fluorescent polymer nanoparticles

Shape and morphology-controlled polymer nanoparticles (Nps) are particularly interesting in various biological applications, such as in phagocytosis and targeted drug delivery [40–42]. The size of the nanoparticles (therapeutic delivery vehicles) affects their movement in and out of the vasculature, whereas the margination of nanoparticles to the vessel wall is driven by their shape [42]. The shape of the nanoparticles can easily be controlled in the case of the crystalline materials because it is driven by facet-specific growth [43]. Oppositely, the polymer possesses an amorphous or semicrystalline nature, and therefore, to control the shape via the bottom-up synthesis at the nanometer-scale level is a highly challenging task. Owing to the advantages of the microfluidic technique such as efficient reactants mixing and fast phase transfer, the control over the shape of the polymer nanoparticles is possible even in a one-step procedure [32].

Flower-shaped polymer nanoparticles (Figure 5A) were obtained when non-ionic polymer polyvinyl pyrrolidone (PVP) was used in the aqueous phase. PVP is well solvated in the water and contributing the effect to solvate the smaller growing hydrophobic nanoparticles in the aqueous medium during ongoing polymerization reaction. The flower-shaped architecture is the result of an aggregation of the multi-aggregates phase, which is dependent on the interface phenomena. The detailed mechanism has been explained in our previous report [33]. The incorporation of a fluorescence dye in the polymer network creates florescence-active nanoparticles. Figure 5B represents the fluorescence spectrum of 1,6-diphenyl-1,3,5-hexatriene (DPH)-doped flower-shaped polymer nanoparticles, which emit the fluorescence light at about 430 nm. On another side, a dumbbell-shaped polymer nanoparticle, as shown in Figure 5C, is obtained when polyelectrolyte poly(sulfonic acid-co-maleic acid) sodium salt (PS-co-PM) was used in the aqueous phase. Formation of the dumbbell-shaped nanoparticles is governed by limited polarization, which is induced by the availability of PS-co-PM at the interface of growing polymer nanoparticles during the ongoing polymerization process at an elevated temperature (97°C). The most preferred form of the nanoparticles is a sphere at minimized interfacial energy. Here, the flexible nature of the PS-co-PM on the particle surface allows a controlled assembling of two differently growing spheres during the desorption phase in the ongoing polymerization process [32]. Finally, the dumbbell-shaped nanoparticles are obtained, which is probably the result of a limited polarization and partial electrostatic repulsion. The concentration of PS-co-PM on the surface of the nanoparticles tunes the surface charge of the obtained nanoparticles together with tuning in the nanoparticle size [32]. The dumbbell-shaped nanoparticles become fluorescence active when 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X) noncovalently bound to the nanoparticle interior, and it emits florescence light at about 535 nm (Figure 5D). Toward the extension of strategy for preparing functional nanoparticles on the basis of size, surface potential, and fluorescence, the composite nanoparticles, which can combine the properties of two or more different domains, were prepared. Dumbbell-shaped polymer nanoparticles show fluorescence spectra at one wavelength with the availability of a specific surface charge, which initiate electrostatic interaction to bind the oppositely charged spherical fluorescent polymer nanoparticles of another fluorescence wavelength. Figure 5E and F shows that negatively charged NBD-X-doped dumbbell nanoparticles binds the 6-propionyl-2-dimethylaminonaphthalene (prodan)-doped positively charged spherical nanoparticles on the surface. By varying the size and density of the spherical nanoparticles on the surface of the dumbbell, a broad library of size, shape, and surface potential-tuned fluorescent composite polymer nanoparticles can be prepared, which is particularly interesting to tailor the mixed properties in a single polymeric system.

Figure 5:

(A) and (B) are the SEM image of DPH (dye)-doped flower-shaped polymer nanoparticles and their fluorescence spectrum, respectively. (C) and (D) are the SEM image of NBD-X (dye)-doped dumbbell-shaped polymer nanoparticles and their fluorescence spectrum, respectively. (E) and (F) are the SEM image of the electrostatic dumbbell-sphere composite polymer nanoparticles and their fluorescence spectrum, respectively, where dumbbell-shaped nanoparticles contained NBD-X dye in the interior, and prodan dye was embedded in the spherical nanoparticles.

3.4 Fluorophore at the surface of nanoparticles

It is required to have the charges on the nanoparticle surface for many of their efficient use such as binding with the targeted site for diagnostic and detection purposes as well as on the functional surface [22]. A demand for preparing the functional nanoparticles is to obtain them in a minimum number of preparation steps and consuming minimum types of reactants. Interestingly here, the aqueous phase is made up of the amphiphilic fluorescence dye (chemical structure shown in Figure 6A, Titan yellow), which mimic the function of the surface active agent. It acts as a surface-stabilizing agent for controlling the particle size and surface potential together with the hierarchical variation in the fluorescence intensities of the nanoparticle in a one-step synthesis. As shown in Figure 6C, the size of the PMMA nanoparticles decreased from about 200 nm down to 80 nm when the concentration of Titan yellow in the aqueous phase increased from 0.01 mm up to 1 mm. It is believed that the diffusion rate of the monomers during the ongoing polymerization is higher when the surface layer of the Titan yellow is weak (lower concentration), and therefore, the obtained particles are of bigger size. However, the mechanism is not clear about how the Titan yellow in the aqueous phase is able to tune the size of the nanoparticles very uniformly. Figure 6D shows that the surface potential of the nanoparticles of about -42 mV was obtained when 1 mm of Titan yellow was used in the aqueous phase. The surface potential of the nanoparticles is gradually decreased down to -10 mV along with the decrease in Titan yellow concentration from 1 mm down to 0.01 mm. At the end of the polymerization reaction, the Titan yellow molecules were irreversibly adsorbed on the surface, and PMMA nanoparticles becomes fluorescence active. As shown in Figure 6B, the Titan yellow-covered PMMA nanoparticles emit the fluorescence light at 425 nm. Obviously, the fluorescence property is not determined exclusively by the fluorescence intensity of the particles, but the fluorescence intensity increases when the surface potential of the obtained nanoparticles also increases with gradual decreasing in particle size simultaneously. Such intensities can impact strongly on the final fluorescence outcome. Hence, the tuning in the size, surface potential, and fluorescence intensity exclusively depends on the concentration of the charged fluorophore in the aqueous phase. Smaller-sized nanoparticles of about 80 nm are highly fluorescent because of higher density of the Titan yellow on the surface. These fluorescence-active homogeneous PMMA nanoparticles with specific surface potentials can be used for many applications as a photonic crystal, to make the monolayer of the fluorescent polymer nanoparticles on a charged sheet among the others.

Figure 6:

(A) SEM images of the obtained PMMA nanoparticles when different concentrations of the Titan yellow have been used in the aqueous phase, (1) 0.1 mm and (2) 0.025 mm, and (3) a chemical structure of Titan yellow. (B) The fluorescence spectra of the PMMA nanoparticles obtained when different concentrations of Titan yellow were used in the aqueous phase. Graphical results of the (C) size and (D) surface potential of the obtained nanoparticles when different concentrations of Titan yellow were used in the aqueous phase.

3.5 Surface functionalization with polypeptides, biomolecules, and fluorophores

Surface functionalization of the particle-based fluorophore is the precondition to use them as an efficient fluorescence label, imaging tool, and targeted reporter or sensor [7]. The detection of the targeted site by labeling and sensoric materials relies on the fast, sensitive, and reproducible interaction [44]. Here, the particle-based fluorophore is in the account, and therefore, the particle homogeneity and surface functional groups are important for systematic outcome and uniform interaction with analytes [45]. For such purpose, a layer-by-layer surface functionalization approach has been applied. Initially, 160 nm-sized CTAB-covered nanoparticles (ζ potential: +20 mV) were prepared (Figure 7A). CTAB molecules can attach irreversibly on the nanoparticle surface where the hydrophilic charged part (-NH₃⁺ functional group) remains free. In next step, therefore, the carboxylate groups of the poly-l-glutamic acid (PGA) interact with nanoparticle surface via electrostatic interaction. The purpose of using polypeptide for surface modification is to having accessibility of free charged ions for further modification. Functionalized PMMA nanoparticles become negatively charged (ζ potential about -10 mV) after modification with PGA. Various types of polyelectrolytes can be used for building the functional layers [46–49], and due to the biocompatibility as well as strong binding affinity through electrostatic interaction, the PGA-PLL assembly is well known. The surface potential of the PMMA nanoparticles again becomes positive (about +40 mV) after the second modification step with cationic polypeptide (poly-l-lysine (PLL)) as shown in Figure 8A. The layer-by-layer (LbL) [50–52] assemblies of PGA and PLL up to the desired number of layers and their thickness are routinely utilized on the surface of the planer sheet and film for the biocompatible purpose to interact with living cells [53–56]. As both PGA and PLL are polypeptides, they have a tendency to interact with the cell surface and with other biomolecules. However, it was claimed that the adhesion force of the cell surface on the PLL-terminating film was higher, whereas no cellular adherence was found on the PGA-ending film in the serum-free medium [57].

Figure 7:

(A) SEM image of the dye-labeled streptavidin-functionalized PMMA nanoparticles. (B) Schematic of the layer-by-layer surface modification via electrostatic, covalent, and secondary binding by biotin-streptavidin coupling. (C) Chemical structures of the PGA, PLL, and NHS-biotin, used for the surface functionalization of the nanoparticles. The camera pictures of the three glass vials, which possess biotin-covered particles, dye-labeled streptavidin-covered particles, and only dye-labeled streptavidin solution, respectively: (D) without UV light and (E) with irradiation of UV light.

Figure 8:

(A) Graphical results of the nanoparticle ζ potential after layer-by-layer surface modification. (B) Fluorescence spectra of PMMA nanoparticles after functionalization with different concentrations of dye-labeled streptavidin.

To study the biological processes such as cell reporters and labeling of the targeted site, biotinylation is widely used in which biotin interacts with active targeted sites [58, 59]. The process of biotinylation is rapid and specific due to the small size of the biotin molecules (244 Da), and therefore, it can be conjugated to many proteins without altering their biological activities. Biotin is a naturally occurring vitamin and binds to the avidin or streptavidin proteins with very high affinity [60]. Biotin possesses a carboxylic acid functional group or active ester group (if it is -NHS functionalized biotin). Therefore, it strongly interacts with the amine group of PLL on the surface of the PMMA nanoparticles through covalent bond. N-Hydroxysuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-biotin (Figure 7C) enables efficient labeling of amine-containing particles or macromolecules. Therefore, PLL-capped PMMA nanoparticles were dispersed in the PBS buffer solution of sulfo-NHS-LC-LC-biotin (sulfosuccinimidyl-6-(biotinamido)-6-hexanamido hexanoate) for strong covalent binding. The sulfo-NHS-ester reagent is water soluble, enabling reaction to be performed in the absence of an organic solvent such as DMSO or DMF. The interaction was confirmed by ζ potential measurements after several washing cycles of the nanoparticles. The surface potential of the biotin-tagged polymer nanoparticles become highly negative (about -40 mV), which confirms the strong covalent binding of the PLL-biotin on the surface of the PMMA nanoparticles at a longer distance (Figure 8A).

On the other hand, biotin has a strong affinity to linked with streptavidin through secondary interaction [59]. Moreover, this interaction of biotin with streptavidin is considered as one of the strongest noncovalent binding (protein-ligand binding), and therefore, it is widely used for many biotechnological applications such as biochemical assays because this particular interaction is attained with high specificity and high uniformity [61]. Biospecificity of the interaction is similar to the antibody-antigen or receptor-ligand recognition, but on a much higher level with respect to the affinity constants. The “long-chained” sulfo-NHS-LC-LC-biotin and sulfo-NHS-biotin both have the same chemical functionality. However, the sulfo-NHS-LC-LC-biotin (Figure 7C) provides a longer spacer arm that is designed to overcome any steric hindrances that may be present between the labeled PMMA particles and streptavidin. To make the entire PMMA nanoparticle network fluorescence active, therefore, dye-labeled streptavidin has been applied to the biotin-covered particles. As biotin-streptavidin interaction is neither covalent nor electrostatic, the surface potential was not affected much; however, it slightly moved to the upward direction (about -30 mV as shown in Figure 8A) after interaction with the dye-labeled streptavidin. We have used AlexaFluor 594 conjugate (dye-labeled streptavidin) of which absorbance maxima is at 590–595 nm and its fluorescence emission at 619 nm. Biotinylated PMMA nanoparticles were incubated in the PBS buffer solution (pH 7.4) of 0.1 mg/ml dye-labeled streptavidin. The PMMA nanoparticles appear completely sky blue after functionalization with the dye-labeled streptavidin (Figure 7D) and showing intense red color in UV exposition (Figure 7E). The blue-colored PMMA nanoparticles emit the fluorescence light at about 619 nm (Figure 8B). In general, immobilization of the biomolecules directly on to the surface of the nanoparticle may not be suitable for many of the biosensors because it leads to low coverage of the biomolecules with reduced activity and nonspecific adsorption. Various surface chemistries, which provide the desired chemical properties for stable and defined binding of ligands, provide high functionality in conjugation with minimum nonspecific binding to the surface have been developed.