A label-free and sensitive fluorescence assay for hyaluronidase activity through electrostatic-controlled quantum dots self-assembly

Polycation-induced CdTe QDs fluorescence quenching

The quantum dots were prepared according to the literature description. ⁴⁷ In a buffer solution (5 mM Tris-HCl, pH = 7.4), the CdTe QDs emit intense blue luminescence and the maximum fluorescence emission is at 534 nm (Figure 2).

OPEN IN VIEWER

Figure 2.

Fluorescence emission spectra of the CdTe QDs (50 nM).

Polycation 1 induced aggregation of CdTe QDs. Figure 3(a) shows that the CdTe QDs emission intensity decreased gradually on increasing the polycation 1 concentration. As reported previously,^48,49 the aggregation of QDs caused the red-shift and quenching of the QDs’ fluorescence by electronic energy transfer between neighboring dots in the QDs aggregate. Figure 3(b) shows that the quenching efficiency (QE) of the QDs increased with the concentration of polycation 1 (0–0.4 µM). Further increasing the polycation 1 concentration caused hardly any change of fluorescence QE, which indicated that the binding of the QDs to the polycation 1 was gradually saturated. This result indicates that polycation 1 could induce aggregation of the QDs and the monomer fluorescence of the QDs was quenched.

OPEN IN VIEWER

Figure 3.

(a) Emission spectra of the CdTe QDs at 534 nm against polycation 1 concentration (0–1.2 µM). (b) Emission intensity changes of the CdTe QDs at 534 nm against polycation 1 concentration (0–1.2 µM).

Disaggregation of the QDs in the presence of HA and fluorescence recovery

Figure 4 shows how the fluorescence spectrum and emission intensities of the CdTe QDs change with increasing amounts of HA, indicating that competitive binding of the negatively charged HA to polycation 1 leads to the liberation of the CdTe QDs. When 1 nM of HA was introduced, the fluorescence recovery of the CdTe QDs reached its maximum. A further increase in HA concentration caused no further increase in the fluorescence intensity, indicating that all of polycation 1 had bound to HA. The fluorescence recovery of the CdTe QDs is directly related to the amount of HA added, which forms the basis for HAase activity monitoring.

OPEN IN VIEWER

Figure 4.

(a) Emission spectra of the QDs against HA concentration (0, 0.005, 0.2, 1, 4, and 10 nM, respectively). (b) Plot of the emission intensity of the CdTe QDs versus HA concentration (0, 0.005, 0.2, 1, 4, and 10 nM, respectively).

The scanning electron microscope (SEM) images of the CdTe QDs sample only, of the CdTe QDs mixed with the polycation 1, and of the CdTe QDs mixed with polycation 1 and HA were recorded. Figure 5 shows that the CdTe QDs changed from a dispersed situation (Figure 5(a)) to aggregation (Figure 5(b)) and the disaggregation of the CdTe QDs in the presence of polycation and HA (Figure 5(c)). This result demonstrates that the positively charged polycation 1 could induce aggregation of the negatively charged CdTe QDs and the intense binding of HA to the polycation induces the disaggregation of CdTe QDs in the presence of HA.

OPEN IN VIEWER

Figure 5.

SEM images of (a) CdTe QDs, (b) CdTe QDs + polycation 1, and (c) CdTe QDs + polycation 1 + HA.

Hyaluronidase activity assay

Figure 6 shows that the CdTe QDs emission intensity gradually decreases with increasing the hyaluronidase enzymatic reaction time (0–50 min) in the presence of 0.25 U/mL HAase. This result indicates that HA is gradually degraded into fragments at an enzymatic reaction time of 50 min and the released polycation 1 could induce the reaggregation of the CdTe QDs.

OPEN IN VIEWER

Figure 6.

Emission intensity of the QDs in the presence of 0.25 U/mL hyaluronidase at different enzymatic reaction times (0–50 min).

Figure 7 shows that the fluorescence spectrum and fluorescence emission intensity of the CdTe QDs decreases with HAase concentration added to the samples at 0–0.8 U/mL. The inset shows a linear relationship between the fluorescence emission intensity and HAase concentration in the range of 0–0.1 U/mL, with a calibration curve of y = 136.510 − 491.32x. The detection limit is as low as 0.01 U/mL (3σ/slope, correlation coefficient R² = 0.989). Compared with previously reported methods,^36–43 our assay method is highly sensitive.

OPEN IN VIEWER

Figure 7.

(a) Emission spectra of the QDs against hyaluronidase concentration (0, 1, 5, 10, 15, 25, 50, and 80 mU/mL, respectively). (b) Plot of the QDs emission intensity at 534 nm versus hyaluronidase concentration at a reaction time of 40 min. Inset: the expanded linear region.

Selectivity studies

The selectivity of our method was studied. Several potential interfering enzymes such as collagenase, ALP, trypsin, and lysozyme were selected to perform the control experiment. Each enzyme was tested under the same experimental conditions as mentioned above. From Figure 8, none of these enzymes brought about the obvious fluorescence signal change of the QDs, which indicated that these enzymes did not interfere with our assay. Thus, our assay is highly selective for HAase.

OPEN IN VIEWER

Figure 8.

Selectivity study. The QDs emission intensity at 543 nm after the addition of different enzymes. Enzyme concentration: 0.25 U/mL.

Assay in biological fluid

Our assay was also tested in a complex mixture sample including 1% human serum. The assay was carried out under the same experimental conditions. Figure 9 shows that the QDs emission intensity decreased gradually with HAase concentration (0, 0.03, 0.15, 0.25, 0.4, and 0.6 U/mL, respectively). This result shows that our method could be applied in a complex biological fluid.

OPEN IN VIEWER

Figure 9.

The QDs emission intensities in the presence of different concentrations of hyaluronidase in 1% human serum added to the assay solution. Columns 0–5: with the addition of 0, 0.03, 0.15, 0.25, 0.4, or 0.6 U/mL HAase, respectively.

Apparatus

A Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc., USA) was used for the fluorescence emission spectra measurements. The excitation wavelength was 350 nm. Excitation and emission slit widths of 3 nm were selected. Quartz cuvettes with a path length of 10 mm were used for fluorescence emission measurements. A Sigma 300 field-emission electron microscope (Carl Zeiss, Jena) was used to acquire SEM images. Unless otherwise specified, all spectra were taken in 5 mM Tris-HCl buffer solution (pH = 7.4).

Materials

Cadmium(II) chloride (CdCl₂·5H₂O) was provided by Beijing Chemical Works (Beijing, China). Tellurium powder and sodium borohydride (NaBH₄) were purchased from the Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 3-Mercaptoacetic acid was purchased from the Sinopharm Chemical Reagent Co., Ltd. Poly(diallyldimethylammonium chloride) (polycation 1; 35 wt% in H₂O; MW <100,000) was purchased from Sigma Aldrich (St. Louis, MO, USA). The polycation 1 concentration is defined as the concentration of the repeating unit. HA, hyaluronidase, lysozyme, and collagenase were supplied by the Sangon Biological Engineering Technology & Service Co., Ltd (Shanghai, China). Trypsin was purchased from the Beijing Dingguo Changsheng Biotech Co., Ltd (Beijing, China). ALP was supplied by the TaKaRa Biotechnology Co., Ltd (Dalian, China). Enzyme solutions were kept at 4 °C before use. The other reagents were used with no further purification and were of analytical grade. Ultrapure water (18.2 MΩ.cm) in all the experiments was prepared with a Milli-Q AE10 filtration system (Millipore, Billerica, MA, USA).

Polycation 1-induced CdTe QDs aggregation and fluorescence quenching

Different amounts of polycation 1 (0, 0.1, 0.2, 0.4, 0.7, 1, and 1.2 µM, respectively) were mixed with buffer solutions containing 4 µL of CdTe QDs (5 µM). The sample solutions were mixed briefly and fluorescence emission spectra and emission intensities at 534 nm were obtained. Total sample volume is 400 µL, buffer is 5 mM Tris-HCl, and pH is 7.4. The QE is defined as: QE = (I₀ − I)/I₀, in which I and I₀ are the emission intensities of the QDs at 534 nm in the presence or absence of polycation 1, respectively.

Fluorescence recovery of the CdTe QDs in the presence of HA

Different amounts of HA and 4 µL of polycation 1 (40 µM) were left standing at room temperature for 5 min and 4 µL of CdTe QDs (5 µM) was introduced. Then, the sample solutions were mixed briefly and the fluorescence emission spectra and emission intensities of the CdTe QDs at 534 nm were obtained. Total sample volume is 400 µL, buffer is 5 mM Tris-HCl, and pH is 7.4.

Assay for hyaluronidase activity

A total volume of 200 µL of buffer solutions containing different concentrations of HAase (0, 0.01, 0.05, 0.1, 0.15, 0.25, 0.5, and 0.8 U/mL) and 4 µL of HA (100 nM) were left standing at 37 °C for 40 min and 4 µL of polycation 1 (40 µM) was introduced. Next, the solution was left standing at room temperature for 5 min and 4 µL of the CdTe QD (5 µM) was introduced. Finally, the sample solution was mixed briefly and the fluorescence emission spectra and emission intensities of the CdTe QDs at 534 nm were obtained. Total sample volume is 400 µL, buffer is 5 mM Tris-HCl, and pH is 7.4.

Selectivity studies

Collagenase, ALP, trypsin, lysozyme, and HAase (0.25 U/mL, each) were added to the buffer solutions containing 4 µL of HA (100 nM), respectively. The sample solutions were kept at 37 °C for 40 min and 4 µL of polycation 1 (40 µM) was introduced. Next, the solutions were left standing at room temperature for 5 min and 4 µL of CdTe QD (5 µM) was introduced. Finally, the sample solution was mixed briefly and fluorescence emission spectra and emission intensities at 534 nm were obtained. Total sample volume is 400 µL, buffer is 5 mM Tris-HCl, and pH is 7.4.

Assay in biological fluid

A total volume of 200 µL of the buffer solutions containing different concentrations of HAase (0, 0.03, 0.15, 0.25, 0.4, and 0.6 U/mL), 4 µL of HA (100 nM), and 1% human serum were prepared. The solutions were kept at 37 °C for 40 min and 4 µL of polycation 1 (40 µM) was introduced. Next, the sample solution was left standing at room temperature for 5 min and 4 µL of CdTe QD (5 µM) was introduced. Finally, the sample solution was mixed briefly and the fluorescence emission spectra and emission intensities at 534 nm were obtained. Total sample volume is 400 µL, buffer is 5 mM Tris-HCl, and pH is 7.4.