F 4600

The dityrosine content of MP was determined as reported by Nikoo, Benjakul, and Xu [21 (link)] with slight modifications. Briefly, each of the MP sample solutions was diluted to 5 mg/mL, and the fluorescence intensity of each sample was measured using a fluorescence spectrophotometer (F-4600, Hitachi High Technologies Corporation of Japan, Tokyo, Japan). The excitation wavelength was 325 nm, the emission wavelength was 420 nm, and the slit width was 10 nm. The results were expressed as an arbitrary unit (a.u.). Each experimental group was measured three times.

Cell growth was monitored by measuring the different absorbance at 680 nm and 750 nm with a UV/VIS spectrophotometer (UV-2802PC UNIC, Shanghai, China), and the dry cell weight was calculated through equation (5)30 (link).

The specific growth rate of H. pluvialis was calculated through equation (6).

where N_t is the dry cell weight at the cultivation of t days (g/L), N₀ is the initial dry cell weight (g/L).
The protein content in H. pluvialis was determined by the dye-binding method as described by Bradford42 (link). ATP concentration was determined lumiically with the spectrometer as described by Mahro43 and the result was presented as μmol per g protein. ROS was measured by reagent of DCFH-DA with fluorescence spectrophotometer (F-4600, Hitachi High-Technologies Corporation, Tokyo, Japan). The excitation wavelength was set at 500 nm and the emission wavelength was set at 525 nm44 (link). The amount of ROS was presented as fluorescence intensity per g protein.

The dityrosine content in the MP sample was determined using a previous method [15 (link)] with a slight modification. Briefly, 0.6 mol/L NaCl was used to dilute the MP sample to 1 mg/mL and the dityrosine fluorescence intensity in the solution was evaluated by a fluorescence spectrophotometer (F-4600, Hitachi High Technologies Corporation, Tokyo, Japan) at the excitation and emission wavelength of 325 and 420 nm, and the slit width of 10 nm, with three measurements for each sample in triplicate. The fluorescence intensity result was defined as the MP dityrosine content in the unit of A.U.

To quantitatively distinguish the target-specific product from the non-target-specific products in CRISDA reactions, PNA invasion-mediated fluorescence measurements were performed by incubating the CRISDA reaction with a biotin-labeled PNA and a Cy5-labeled PNA probe (both at 100 nM) targeting the middle region of the amplicon, which was subject to magnetic pull-down and fluorescence measurements. Briefly, 2 μL of the PNA mixture (biotin-labeled PNA and Cy5-labeled PNA both at 1 μM) were added to the 20 μL of CRISDA reaction. The mixture was incubated at ambient temperature or 37 °C for 15 min. Afterwards, 3 μL of streptavidin-coated magnetic beads (Dynabeads™ MyOne™ Streptavidin C1) were introduced and the mixture was further incubated at ambient temperature or 37 °C for 15 min. Subsequently, the complex containing the specific amplicon and two PNA probes was isolated from non-specific products through magnetic pull-down. The beads were resuspended in 100 μL of the reaction buffer supplemented with 0.4% SDS and incubated at room temperature for 15 min. The fluorescence intensity of the supernatants was determined on a fluorescence spectrophotometer F-4600 (Hitachi High-Technologies Corporation). In each replicate, fluorescence intensities of CRISDA reactions with various target concentrations were normalized against the one containing the highest target concentration.

The surface hydrophobicity of gluten samples was measured according to the method of Li, et al. [29 (link)] with some modifications. Briefly, gluten (400 g) was introduced to 20 mL 0.01 M phosphate buffer (pH 7.0) and stirred for 2 h, followed by centrifuging 8000× g for 10 min. The 4 mL diluted sample was mixed with 50 μL 8-aniline-1-naphthalene sulfonic acid (ANS) and its fluorescence intensity was measured in a fluorescence spectrophotometer (F-4600, Hitachi, Japan). The excitation and emission wavelengths are 370 nm and 490 nm, respectively, and the slit width is 5 nm. The surface hydrophobicity of gluten corresponds to the slope of fluorescence intensity to protein concentration.

A PBS solution with pH value of 7.4 was prepared by adding 5.8032 g Na₂HPO₄·12H₂O and 0.8894 g NaH₂PO₄·2H₂O into 1000 mL distilled water. A NIR laser was fixed 15 cm higher than the center of the scaffolds. Under the irradiation of NIR (λ = 808 nm, 4.6 W/cm²), the temperatures of the media with the scaffolds were detected by thermocouple thermometer (Brannan, Cleator Moor, England). The NIR-light-triggered drug release tests of MCSC 1:3/DOX and MCSC 1:7/DOX scaffolds were performed by the immersion of DOX loaded scaffold in 5 ml PBS solution under orbital shaking of 80 rpm. In brief, the probe of NIR laser (λ = 808 nm, 4.6 W·cm⁻²) was fixed 15 cm higher from the center of the scaffolds. At given time intervals, the scaffolds were irradiated by NIR laser for 6 min. Then 1.0 ml DOX-release medium was extracted and supplemented with 1.0 ml fresh PBS. The DOX concentrations of the DOX-release medium were characterized by fluorescence spectrophotometer (F-4600, Hitachi, Tokyo, Japan) using a xenon lamp as excitation source (λ = 495 nm). The in vitro drug release of the MCSC 1:7/DOX or MCSC 1:3/DOX scaffolds without NIR irradiation was performed with similar procedures.