2.1.

Chemicals and Materials

Silicon tetrabromide (${\mathrm{SiBr}}_4$), tetraoctylammoniabromide (TOAB), lithium aluminum hydride (${\mathrm{LiAlH}}_4$, 1.0 M THF solution), 1,7-octadiene, Hoescht 33258, and $10\times$ trypsin EDTA (1 mM) stock were purchased from Sigma-Aldrich and were used as received unless otherwise stated. RGDC peptide was purchased from Genscript. The fluorescent dye DY485-NHS was obtained from Dyomics. Dry solvents were obtained by passing through a PURE-SOLV (Innovative Technology) solvent purification system with water content below 15 ppm.

2.2.

Synthesis and Purification of 1,7-Octadiene-Passivated Silicon Quantum Dots

Hydrogen-terminated SiQDs were prepared in accordance with a published method.^8,25–27 In brief, all experiments were performed under Ar using a standard Schlenk line setup. In a typical experiment, $100\mu \mathrm{L}$ of ${\mathrm{SiBr}}_4$ ($0.8\times {10}^{-3}\text{mole}$) and 1.5 g TOAB ($2.7\times {10}^{-3}\text{mole}$) were dissolved in 100-mL toluene in a 250-mL three-neck flask. The mixture was sonicated for 20 min. An excess amount of ${\mathrm{LiAlH}}_4$ ($6\times {10}^{-3}\text{mole}$, excess) was then added, and the mixture was further stirred and sonicated for 45 min. The reaction was then quenched with dry ethanol until no bubbles were seen and kept under argon.

Alkene-terminated SiQDs were prepared by reacting 1,7-octadiene with hydrogen-terminated SiQDs and then purified via size exclusion chromatography. The hydrogen-terminated SiQDs obtained in the previous step were transferred to a 250-mL quartz reaction vessel (ACE Glass Inc.) via a cannula needle under Ar. For each hydrosilylation reaction, 4 mL of octadiene was added, and the mixture was treated with UV (254 nm) for 15 h. After the reaction, all solvent and unreacted alkene were evaporated under reduced pressure and elevated temperature, with the crude product obtained as pale-yellow oil. Then water ($3\times 20\mathrm{mL}$) and hexane ($3\times 20\mathrm{mL}$) were added to the mixture. The hexane layer was extracted and was passed through a PVDF membrane with $0.45\text{-}\mu \mathrm{m}$ pore size. To further purify octadiene-passivated SiQDs, size-exclusion chromatography was used. All of the hexane was evaporated and particles were resuspended in 3 mL of toluene. Bio-Beads SX bead was used as the stationary phase and toluene was used as the eluent with no pressure applied. Only fractions that showed blue photoluminescence (PL) under a UV lamp were collected. The column was washed with twice the amount of the beads’ volume after purification.

2.3.

Preparation of SiQDs-DY485 FRET Conjugates

For a typical thiol-ene “click” reaction on the alkene-functionalized SiQDs, the solvent containing 5 mg of SiQDs-octadiene dispersion was first evaporated, and then 1 mL of peptide (sequence: N’-RGDC-C’) DMSO or DMF solution at concentration of $5\mathrm{mg}/\mathrm{mL}$ was added. The mixture was treated under UV ($4\times 8\mathrm{W}$) for 5 h to allow sufficient coupling of the thiol-ene click reaction to complete. The mixture was then dialyzed with Tube-O-Dialyzer (1.5k MWCO, G-Biosciences) in $1\times$ phosphate buffer saline (PBS) at 4°C for 24 h with the buffer replaced every 8 h. The sample was then recovered, and DY485-NHS was added in the desired concentration for 3 h to allow the coupling reaction to complete.

2.4.

Characterization of Morphology and Optical Properties of SiQDs

The size and morphology of the synthesized SiQDs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements. TEM measurements were performed on a Philip CM 200 microscope operated at 200 kV. All TEM images were visualized without staining. DLS measurements were performed on a Brookhaven 90 Plus Instrument. Measurements were performed at 25°C and intensity mean was plotted versus the size distribution. Experimental parameters used are as follows: scan time: 50; buffer condition: hexane for hydrophobic surface nanoparticles and PBS for hydrophilic surface nanoparticles; and scattering angle: 173 deg. Surface functionalization of SiQDs was characterized by FT-IR spectroscopy. FT-IR measurements were performed on an Avatar 320 FT-IR spectrometer. Measurements were done by dropping $10\mu \mathrm{L}$ of concentrated SiQD dispersion onto a pellet premade by grinding a powder of KBr. H-NMR measurements were performed on a Bruker 300 MHz instrument to confirm the purity of synthesized nanoparticles. The optical properties of SiQDs were characterized by UV–vis and fluorescence spectroscopy. UV–vis spectra were recorded on a Cary-50 UV–vis spectrometer and fluorescence spectra measured on a Cary-Eclipse fluorescence spectrometer.

3.1.

Characterization of Surface-Modified Silicon Quantum Dots

The SiQDs were characterized extensively prior to utilizing the SiQD-RGDC-DY485 biosensors. TEM results indicated that the obtained nanoparticles were spherical in shape and relatively monodispersive with an average size of the silicon cores of $3.4\pm 0.7\mathrm{nm}$ [Fig. 2(a)]. High-resolution TEM images revealed lattice fringes matching the (220) lattice spacing of silicon. DLS measurements showed that the hydrodynamic size of the SiQDs was $5.4\pm 0.6\mathrm{nm}$ [Fig. 2(b)]. After conjugation of the peptide, there was a slight increase in SiQDs size, with the small peak measured as $7.2\pm 1.2\mathrm{nm}$ [Fig. 2(c)]. Greater amounts of aggregation were also observed after the modification process. For successful fabrication of the SiQDs FRET donor, we highlight the importance of nanoparticle purification. After each surface modification step, nanoparticles showed expected signatures with FT-IR measurements [Fig. 2(e)]. In particular, bands at 1600 at $3055{\mathrm{cm}}^{-1}$ were due to the distal alkene on the surface, whereas peaks at 3400 and $1700{\mathrm{cm}}^{-1}$ were evidence for the successful grafting of the peptides. With the protocol used, typically $\sim 10\mathrm{mg}$ of purified SiQDs were obtained, while adding more precursors led to aggregated by products and particles were no longer monodisperse.

Fig. 2

Characterization of size and surfaces of SiQDs used as the FRET donors. (a) Representative TEM images and high-resolution (HR-TEM) images showing the spherical morphology and lattice fringes of nanoparticles synthesized (b, c) DLS measurements confirming the narrow size distribution of (b) alkene and (c) peptide conjugated SiQDs. (d) Picture showing the fluorescence of the nanoparticles in different phases obtained from each surface conjugation step. The nanoparticles were dispersed in solvents containing hexane (top layer) or Milli-Q water (bottom layer) under direct excitation using a 365-nm UV lamp and (e) FT-IR of surface modified SiQDs. (f) Spectral overlay between SiQDs and DY485 as also indicated in Ref. 28.

The progress of the surface modification could also be monitored by the solvent phases in which the SiQDs could be dispersed and from their luminescence [Fig. 2(d)]. When modified with 1,7-octadiene, the nanoparticles were only dispersible in nonpolar solvents such as hexane and naturally only the luminescence of the SiQDs was observed. After attachment of the hydrophilic peptide onto the surface, the SiQDs became dispersible in water. Further modification with dye molecules resulted in the SiQD-RGDC-DY485 sensor remaining in aqueous solution but now the luminescence changed from blue to orange upon UV excitation of 365 nm. This color change reflected FRET from the SiQD donor to the DY485 acceptor with its characteristic emission. The optical characteristics of the SiQD-RGDC-DY485 sensors were described in a conference proceedings, as is shown in Fig. 2(f).²⁸

The organic dye acceptor used in this study, DY485, was chosen primarily because it has a large Stokes shift of $\sim 85\mathrm{nm}$ [Fig. 2(f)], which provides minimal overlap between the emissions of the SiQD donor and the acceptor. Hence, emission from a donor channel can be observed without interference from the acceptor. Furthermore, the absorption of DY485 exhibits good overlap with the emission peak of SiQDs as needed for a good FRET donor/acceptor pair.¹³ With the donor–acceptor pair presented herein, the Förster radius was calculated to be $\sim 3.2\mathrm{nm}$, where the spacing between the dye and the nanoparticle was estimated to be $\sim 2.5\mathrm{nm}$. This calculation was determined from the surface of the SiQDs as emission from SiQDs has been previously shown to most likely be interface dominated.^22,27–29

3.2.

Sensor Performance

Coupling the dye to the SiQDs resulted in FRET occurring as shown visually in Fig. 2(d). The spectral changes of the SiQD-RGDC-DY485 as a function of time as the acceptor dye is attached are shown in Fig. 3(a). What is apparent from this figure is that the growth on the acceptor emission as a function of time as expected for with more dye being attached with longer coupling times. Less expected was the only minor decrease in the donor peak during the coupling reaction. A closer inspection for the PL trend at the donor channel [Fig. 3(b)] showed that PL intensity ratio dropped immediately after the addition of the dye, where after the intensity remained fairly stable with less than 10% variation over the remaining reaction time. We attribute this observation to two factors. The first being that silicon is an indirect bandgap semiconductor as in contrast to conventional quantum dots which are typically made from direct bandgap materials. This has led to lower quantum yield and also a strong impact of surface properties over photophysics of the particles, as seen by us and other groups previously.^12,22,30,31 The second factor is that since we did not purify the conjugate after dye coupling, the drop of signal at the donor channel could be predominated by strong absorption of the dye. Note this cannot be attributed to nonspecific adsorption of the dye because a control experiment with hydrolyzed DY485 did not increase the acceptor channel signal [Fig. 3(e)]. The response of the sensor to the addition of trypsin is shown in Figs. 3(b) and 3(c). Upon addition of the trypsin, the FRET efficiency to the accept dye decreased as indicated by the decrease in intensity of the emission peak at 550 nm. This result suggests that trypsin has cleaved the peptide linkage between the SiQDs and the dye and hence less dye is within appreciable FRET distance. Note again the donor emission peak is insensitive to the extent of FRET. The timescale over which this cleavage occurs is shown in Figs. 3(b) and 3(f). The fact that the sensor is specific toward trypsin is seen in Fig. 3(f) where additional collagenase ($100\mu \mathrm{g}/\mathrm{mL}$) or MMP2 ($140\mu \mathrm{g}/\mathrm{mL}$) under the same experimental conditions resulted in no change in luminescence and hence no cleavage of the peptide linker.

Fig. 3

Sensor performance. (a) Change of PL with time upon addition of DY485 ($20\mu \mathrm{M}$). An immediate drop of PL was seen after the addition of the dye acceptor, while the donor peak remained largely stable throughout the rest of the reaction. Excitation wavelength: 350 nm. (b) Typical $I_{\mathrm{d}}/I_{\mathrm{a}}$ trend monitored over the entire experiment. (c) Change of PL upon the addition of trypsin at $460\mu \mathrm{g}/\mathrm{mL}$. (d) Enzymatic response of the SiQDs protease sensor at different trypsin concentrations. (e) Reaction with hydrolyzed DY485 showed a drop in the donor channel but no increase of intensity in the acceptor channel. (f) Enzyme specificity study using collagenase ($100\mu \mathrm{g}/\mathrm{mL}$) and matrix metalloproteinases 2 (MMP2, $140\mu \mathrm{g}/\mathrm{mL}$) which showed no effect on the FRET signal in contrast to clear change for trypsin ($115\mu \mathrm{g}/\mathrm{mL}$). Experiments were performed with $20\mu \mathrm{M}$ of DY485 with excitation wavelength of 350 nm.

Fig. 4

Enzyme kinetics. The enzymatic response of the SiQDs protease sensor showing the raw data (blue stars) versus fitted rate curves based on a global nonlinear fit to the integrated Michaelis–Menten equations with (a–e) corresponding to the different trypsin protease concentrations shown. The results from the fitting process are also shown in the lower right corner.

3.3.

Kinetic Analysis of Enzyme Activity

For protease sensing, the FRET conjugate was treated with trypsin, which cleaves at the C terminus of glycine of the peptide sequence. With an increasing amount of enzyme added, the respective PL measurements indicated a more significant change of FRET response until a maximum change was reached at $\sim 460\mu \mathrm{g}/\mathrm{mL}$ [Fig. 3(d)]. This was in comparison to the control group treated with PBS, which did not show any appreciable change of PL throughout the experiment; thus confirming the change in FRET response was due to the cleavage of the peptide. The reaction kinetics of the enzymatic cleavage as shown in Fig. 3(d) was then investigated (Fig. 4). With the stepwise surface modification strategy used, the substrate concentration and nanoparticle/ligand ratios were not known.^32,33 For this reason, a modified approach for studying the surface reaction kinetics of our system was needed. To do this we used $I_{\mathrm{d}}/I_{\mathrm{a}}$ as a function of enzyme reaction time, which was then fitted with a global nonlinear regression model using an integrated form of the Michaelis–Menten equation using Lambert function and known enzyme concentration.³⁴

More specifically, protease reactions progress as

With the rate equation given as

Since in our case, the substrate concentration is unknown while the enzyme concentration is known, which is opposite to the situation for conventional studies, the analysis of our data is based on direct integration of the Michaelis–Menten equation using the Lambert $W$ function as shown by Schnell and Mendoza³⁴

Here, $W_0$ stands for the Lambert W equation which can be solved by accurate straightforward numerical approaches via MATLAB^®. The maximum enzymatic turnover frequency, $k_{\mathrm{cat}}$, is simply the maximum rate achieved by the system, $V_{\mathrm{m}}$, divided by ${[\mathrm{E}]}_0$, such that

Here, the total enzyme concentration ${[\mathrm{E}]}_0$ is known, but not the total substrate concentration ${[\mathrm{S}]}_0$. Therefore, by inserting Eq. (4) into Eq. (3), we obtain

An important assumption is made here, such that within the measurements range $I_{\mathrm{A}}$ is a linear function of the substrate density on the surface of SiQDs. This comes from the fact that FRET induced fluorescence changes at the donor channel due to FRET are very small. Indeed, the application of the quasisteady-state approximation to the donor-excited channel gives its concentration expression called $[D^{*}]$ as a function of the total donor concentration $[D]$ and the different rate constants [$k_{\mathrm{exc}}$ for the excitation, $k_F$ for the relaxation by fluorescence emission, $k_{\mathrm{FRET}}$ for the relaxation by FRET to the acceptor, $k_i$ all other relaxation pathway (thermal relaxation and all other quenching processes), and [S] the substrate concentration (product of nanoparticle concentration and number of substrates per nanoparticle)]

As the fluorescence is proportional to the rate of relaxation by fluorescence emission (first-order reaction with $[D^{*}]$ as reactant and $k_F$ as rate constant), the fact that little change in the donor channel is observed as a function of the acceptor concentration suggests that $[A]k_{\mathrm{FRET}}\ll k_{\mathrm{exc}}+k_F+\sum _{i=1}^N{k}_i$. In other words, the donor quenching observed is dominated by factors other than FRET. This lets us rewrite the last expression of the excited donor concentration which is independent of the substrate concentration

Similarly, the excited acceptors’ concentration can be expressed as

If we assume that the intensity in the acceptor channel is proportional to $[S^{*}]$, the independence of $[D^{*}]$ with [S] shows that $I_{\mathrm{a}}$ is proportional to $[S]$. However, a final background value has to be added into the modeling. Using this assumption, we can now relate Eq. (8) directly to the observed ($I_{\mathrm{d}}/I_{\mathrm{a}}$) ratio at any given time ${(I_{\mathrm{d}}/I_{\mathrm{a}})}_{\mathrm{t}}$, assuming a background final value of ${(I_{\mathrm{d}}/I_{\mathrm{a}})}_{\infty }$ and by defining the molar concentration response of ($I_{\mathrm{d}}/I_{\mathrm{a}}$) as $ϵ[(I_{\mathrm{d}}/I_{\mathrm{a}})]$, we obtain

This equation was then used as the basis of a global fit for all five ($I_{\mathrm{d}}/I_{\mathrm{a}}$) curves below.

By this method, an estimate of both $k_{\mathrm{cat}}/K_m=1.06{\mathrm{mM}}^{-1}{\mathrm{s}}^{-1}$, and the total substrate concentration ${[S]}_0=0.48\mu \mathrm{M}$ was obtained. It should be noted that with the method devised it was not possible to obtain independent values of $K_m$ and $k_{\mathrm{cat}}$, but only their ratio as the results suggested $[\mathrm{E}]\gg [\mathrm{ES}]$. The fitting also suggested the value of $k_{\mathrm{cat}}/K_m$ was about three to five times lower than the reported value for trypsin-catalyzed proteolytic cleavage of a longer peptide conjugate on conventional quantum dots.³³

3.4.

Nanoparticle Toxicity

To test nanoparticle toxicity, we performed MTT toxicity experiments with the SiQDs donor and the FRET conjugate in vitro using HeLa cells (Fig. 5). The particles were incubated with the cells overnight at different concentrations, and no apparent toxic effect was seen for particle amounts up to $256\mu \mathrm{g}/\mathrm{mL}$ compared with the control group, where only PBS was applied. The results were consistent with previous reports showing no toxic effect of SiQDs.^26,27,35,36

Fig. 5

Nanoparticle toxicity. MTT toxicity test after 24 h incubation with SiQDs in reference to control group treated with PBS. The results suggest no acute toxicity for nanoparticle concentration up to $256\mu \mathrm{g}/\mathrm{mL}$.