Detecting the acellular oxidative reactivity of nanoparticles

Liza M M Roger, Nastassja Lewinski, Lynn Secondo, Jasmine Wang

Published: 2021-11-16 DOI: 10.17504/protocols.io.bz4jp8un

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Abstract

This protocol was designed to detect acellular oxidative reactivity of nanoparticles

Steps

Materials

Hydrogen peroxide solution (30 wt% in water, Sigma Aldrich cat. no. 216763)
2′,7′-Dichlorodihydrofluorescein diacetate (>97%, Sigma Aldrich cat. no. D6883)

NOTE: Once opened, the solid DCFH-DA must be kept under argon at -20°C.
Methanol (HPLC grade)

NOTE: Can substitute with pure Ethanol 190 Proof (Decon Labs cat. no. V1101)
PBS, 1X Phosphate-Buffered Saline (ThermoFisher cat. no. 10010023)
Horseradish peroxidase (~150 units/mg, 100mg, MW ~40,000 Da, Sigma Aldrich cat. no. 77332)
Sodium hydroxide (10M in water, Sigma Aldrich cat. no. 72068)

DCFH-DA stock solution preparation

Add 24.4 mg DCFH-DA (MW 487.29 g/mol) powder to a 50 mL volumetric flask.
Fill flask to 50 mL volume line with methanol.
This makes a 1 mM DCFH-DA stock concentration.
Wrap flask with aluminum foil.
This stock solution can be stored in the freezer at -20°C for 4 months.

H2O2 stock solution preparation

Add 114 µL of 30 wt% H₂O₂to a 10 mL volumetric flask.
Fill flask to 10 mL volume line with Milli-Q water.
This makes a 0.1 M H₂O₂stock solution.

[This stock solution should be made fresh]

NaOH stock solution preparation

Add 50 µL of 10 M NaOH to a 50 mL volumetric flask.
Fill flask to 50 mL volume line with Milli-Q water.
This makes a 10 mM NaOH stock solution.

[This stock solution can be stored at 22°C for 1 month]

H2O2 working solution preparation

Add 20 µL of 0.1 M H₂O₂stock solution to a 10 mL volumetric flask.
Fill flask to 10 mL volume line with Milli-Q water.

This makes a 200 µM H₂O₂working solution

DCFH working solution preparation

Add 4 mL of 10 mM NaOH and 1 mL of 1mM DCFH-DA stock solution to a 20 mL volumetric flask.
Wrap flask in aluminum foil.
Let the mixture react at room temperature for 30 min.
Quench the reaction by diluting the DCFH-DA in NaOH solution with phosphate buffer saline (1X PBS, pH 7.2-7.4) up to the 20 mL mark on the volumetric flask.

This makes a 50 µM DCFH-DA concentration.

DCFH-HRP working solution preparation

Add 1 mg of horseradish peroxidase (HRP) powder (~150 units/mg) to a 50 mL volumetric flask.
Add 10 mL of freshly prepared 50 µM DCFH solution to the flask.
Fill the flask to the volume line with 1X PBS.

This makes a 10 µM DCFH with 3 unit/mL HRP working solution.

Analyte preparation (for analytes in suspension)

- If the concentration is known: prepare 1 mL of a 100 µg/mL analyte suspension by diluting the analyte stock suspension using 1X PBS as the diluent.

If the concentration is unknown: prepare 1 mL of a 1:10 dilution analyte suspension by adding 100 µL of the analyte stock suspension to 900 µL 1X PBS.

Mix thoroughly using a vortex mixer or bath sonicator.

Analyte preparation (for powder analytes)

Place a 2.5 mL Eppendorf tube inside the static eliminator built into the ultramicrobalance and run to dissipate any electrostatic charge in the plastic.
Weigh 0.2 mg of powder analyte directly into the Eppendorf tube.
Add 0.1 mL of dimethyl sulfoxide (DMSO) to disperse the powder analyte.
Add 1.90 mL 1X PBS.
This makes a 100 µg/mL analyte suspension from powder.

Dosing plate preparation

10.

Using a round bottom 96-well plate, prepare the dosing plate according to the following layouts.

A	B	C	D	E
		H2O	H2O2 Dilutions	Final / Total
Row Number	Chemical Dose	Volume (µL)	Volume (µL)	Volume (µL)
B	0 µM	50	0	50
C	12.5 µM	50	50	50
D	25 µM	50	50	50
E	50 µM	50	50	50
F	100 µM	50	50	50
G	200 µM	0	100	50

		PBS	Analyte Dilutions	Final / Total
Row Number	Analyte Dose	Volume (µL)	Volume (µL)	Volume (µL)
B	0 µg/mL	50	0	50
C	1 µg/mL	90	10	50
D	10 µg/mL	60	40	50
E	25 µg/mL	50	50	50
F	50 µg/mL	50	50	50
G	100 µg/mL	0	100	50

Fluorescence measurement

11.

Using a multipipette, transfer 20 µL from each well in the dosing plate to a black 96-well plate. Note: Rows A & H and Column 12 will not be filled.
Using a 8-channel multipipette, add 200 µL of the DCFH – HRP working solution to wells in Columns 1-3, 5-7, 9-11.
Add 220 µL of the DCFH – HRP working solution to wells in Column 4.
Add 220 µL of 1X PBS to wells in Column 8.
Place the 96-well plate into the multiplate reader (Cytation 3) thermostatted at 37°C and after shaking the platefor 5 seconds, wait 2 minutes thenstart fluorescent intensity measurements.
The fluorescence signal is measured every minute for 60 minutes. The spectroscopic reading occurs at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Note: Data is presented by first background correcting for the DCHF-HRP signal then plotting the signal increase compared to the blank (0 µM or 0 µg/mL) at specific times or through the change in fluorescence response over time at a given concentration.