Conclusion

In this work, a nano-sized particle with silica matrix coated with PEG was fabricated.

The particles were synthesized using a modified Stöber method incorporating PEG monomethyl work as a steric stabilizer to reduce aggregation between the particles while narrow down particle diameter. The particle is also entrapped catalase, [Ru(dpp)3]²⁺ and Oregon Green 488-dextran with retaining enzyme activity. The combination of the catalase and dyes enabled a ratiometric fluorescent determination of hydrogen peroxide in vitro. We also find that the amount of protein in sol-gel compositions are closely concerned with the particle morphology and entrapping rate to fluorescence dyes.

Continued investigations in the future will focus on in vitro measurement. The cell uptake rate by endocytosis and cytotoxicity will be study next. Meanwhile, further improve the detecting limit is also important to let the CAT-PEBBLEs to be able to give real-time hydrogen peroxide detection within physiological concentration (from μM to mM).

In summary, we report details the preparation of enzyme-entrapped PEBBLE nanosensors and their characterization.The same fabrication method can be applied to produce photodynamic nanoplatforms for various applications.

Table 1. The reported PEBBLEs sensors

Matrix material Analytes

Encapsulated components Indicator dye & reference dye

/ Other components

Polyacrylamide pH Ca²⁺

Five varieties of pH-sensitive sensors and three different calcium-selective sensors &

Sulforhodamine 101

Oregon Green 488-dextran or Texas Red-dextran

platinum (II) octaethylporphine ketone &

octaethylporphine

[47]

Polyacrylamide Cu²⁺ Cu⁺ The red fluorescent protein (DsRed) &

Alexa Fluor 488 dextran [48]

1 PDMA: Polydecylmethacrylate; ² PTMS: Phenyltrimethoxysilane

Table 2. Conditions for the synthesis of PEBBLEs

Reaction Composition

Containing EtOH 50%, NH4OH 10%, TEOS 4%, H2O 36%

and PEG 6000 2.5g¹ for each reaction.

2 Catalase from Bovine Liver, powder, 2950units/mg

3 Catalase from Corynebacterium glutamicum, solution, 1500units/μL

410mg, 30mg, 50mg, 80mg and 100mg of Catalase from Bovine Liver were added

Figure 1. SEM images of Silica and Silica-PEG particles

(a) Particle synthesized by silica only, exhibiting a size of 700-800 nm in diameter. (b) Particles generated by silica-PEG exhibit a size distribution of 400-500 nm in diameter. (c) Silica-PEG-[Ru(dpp)3]²⁺ particles exhibit a size of 700-800 nm in diameter. (d)

Silica-PEG-Oregon Green 488-Dextran^® particles exhibit a size of 300-400 nm in diameter.

(a) (b)

(d) (c)

Figure 2. SEM images of Silica-PEG-HRP particles

HRP was used as an enzyme model to fabricate enzyme-entrapped particles. As the image shows, the morphology of Silica-PEG-HRP was irregular and the diameter was also diverse from 200 to 500 nm in diameter. Some agglomerate could also be found in the SEM images. Particle was calibrated by the activity assay and the specific activity was 0.3 units/mg particles. This result revealed that the method was enabling to trap enzyme within.

Figure 3. SEM images of Silica-PEG-bCAT particles

(a) Silica-PEG-bCAT (10 mg) particle. (b) Silica-PEG-bCAT (30 mg) particle. (c) Silica-PEG-bCAT (50 mg) particle. (d) Silica-PEG-bCAT (80 mg) particle. (e)

Silica-PEG-bCAT (100 mg) particle. As the SEM images show, adding proteins (Catalase from bovine liver) into the reaction shows smaller particle size accompany with

morphology changes. When catalase amount increased from 10 mg to 100 mg in the reaction, the particles became irregular and aggregated.

(a) (b) (c)

(d) (e)

Figure 4. SEM images of bCAT-Ru/488-PEBBLEs particles

The generated of bCAT-Ru/488-PEBBLEs exhibited a size in a range of 100-200 nm with a specific activity of 0.03 units/mg particles

Catalase Calibration Curve

(ABTS absorbance V.S. Catalase activity)

0 0.5 1 1.5 2 2.5

3 3.5 4 4.5 5 5.5 6 6.5

Catalase Activity (units)

ABTS Absorbance Value

Figure 5. Catalase calibration curve

Catalase-HRP coupled enzyme assay was carried out to determine the activity of

bCAT-Ru/488-PEBBLE. Since PEBBLEs raises no obvious background in oxidized ABTS solution at 405 nm, this calibration curve is suitable for activity test of

bCAT-Ru/488-PEBBLE, especially when particles with lower specific activity.

Figure 6. SEM images of Silica-PEG-cgCAT (from Corynebacterium glutamicum) particles

(a) Silica-PEG-cgCAT (10 μL) exhibits a particle size of 300-400 nm with a specific activity of 20 units/mg (b) Silica-PEG-cgCAT (30μL) exhibits a particle size of 300-400 nm with a specific activity of 90 units/mg (c) Silica-PEG-cgCAT (50μL) exhibits a particle size of 300-400 nm with a specific activity of 138units/mg. Particles are fabricated by adding different volume of Catalase from Corynebacterium glutamicum (solution, 1500units/μL) and fluorescence dyes. Reaction composition was summarized in Table 2.

(a) (b)

(c)

Figure 7. Fluorescence emission spectrum of bCAT-Ru/488-PEBBLEs

The quenching effect of H2O2 on the fluorescent peak at 607 nm ([Ru(dpp)3]²⁺) indicated that the particle was ready to be used for the detection of hydrogen peroxide.

0.019g particle V.S. H

O

concentration (10min.)

0 200 400 600 800 1000 1200 1400

510 560 610 660 710

Wavelength (nm)

Intensity

reference 20mM 40mM 60mM 80mM

0

510 530 550 570 590 610 630 650 670 690 710 730 750

Wavelength (nm)

Figure 8. Fluorescence spectrum of cgCAT/BSA-Ru/488-PEBBLE v.s. [H₂O₂] Fluorescence spectrum shows that when the ratio of catalase to BSA increased in the reaction composition, the fluorescencedye entrapping efficiency was altered. When the amount of protein increased, the intensity of fluorescent peak at 607 nm ([Ru(dpp)3]²⁺) increased, while the intensity of fluorescent peak at 524 nm (Oregon Green 488-Dextran^®) decreases.

cgCAT/BSA-Ru/488- PEBBLEs

Figure 9. Fluorescence emission spectrum cgCAT/BSA-Ru/488-PEBBLEs.

(a) CAT (30μL)/BSA (2.5mg)- Ru/488-PEBBLEs v.s. [H2O2] fluorescence spectrum. The spectrum shows that the fluorescence intensity of peak at 607 nm ([Ru(dpp)3]²⁺) was inversely proportional to the [H2O2] concentration and capable to detect [H2O2] changes in the environment. (b) Normalized standard curve from data of (a).

(a)

(b)

Figure 10. SEM images of cgCAT/BSA-RU/488-PEBBLEs

(a) The cgCAT/BSA(2.5 mg)-Ru/488-PEBBLE exhibits a particle size of 200-300 nm with a specific activity of 7.8 units/mg particles (b) The cgCAT/BSA(10

mg)-Ru/488-PEBBLE exhibits a particle size of 200-300 nm with a specific activity of

<0.5 units/mg. BSA may increase [Ru(dpp)3]²⁺ entrapping efficiency. The particle prepared in the presence of 2.5mg BSA was smooth on the surface and spherical in shape. In

comparison, particles prepared in the presence of 10 mg BSA, exhibited roughed surface.

(a)

(b)

Figure 11. Fluorescent microscopy images of HeLa cell incubated with cgCAT/BSA-Ru/488-PEBBLEs

As the green fluorescence of the Oregon Green 488-Dextran^® (524nm) showed in Figure 11b, indicated that the fluorescence signal of the cgCAT/BSA-Ru/488-PEBBLEs is successfully entrapped in the silica-PEG matrix and localized inside of the HeLa cell (as pointed by the white arrow in the figure). In contrast, Silica-PEG particle were also incubated with HeLa cell, which showed no fluorescence signals.