To begin, dissolve two milligrams of multivalent PEG with maleimide reactive group, and one milliliter of 100 millimolar phosphate buffer. Filter the solution through a 0.2 micrometer filter. Add the cysteine terminated DNA peptide conjugates to the solution.
Use size exclusion chromatography to remove the unconjugated materials. Run the samples in PBS. Monitor the absorbance of the to detect DNA and peptides.
Place the sample in the centrifugal filter tubes and concentrate the synthesized nanosensors by centrifugation according to the manufacturer's recommended speed. Use a 96-well plate as the base for a customized housing chamber. To collect the urine for baseline measurements, first place a mouse in the housing chamber.
Apply gentle pressure on the restrained mouse's bladder to expel any remaining urine onto the plate. Pipette the collected urine into a 1.5 milliliter tube. Prepare 200 microliters of the nanosensor injection solution in sterile PBS.
Intravenously inject 200 microliters of the solution into each mouse. After one hour of injection, collect the urine samples from the control and experimental mice. For fluorescence-based CRISPR detection of the DNA barcodes, centrifuge the urine samples at 800 G for five minutes at room temperature.
Now combine the CRISPR reagents, then add the Cas12a enzyme before gently pipetting the reaction mixture up and down. Incubate the reaction mixture at 37 degrees Celsius for 30 minutes. Then run the reporter reaction using the FRET-based reporter DNA mixture.
Add the mixture to a 384-well plate and immediately measure the fluorescence at 37 degrees Celsius every two minutes for three hours to monitor the cleavage kinetics. To perform paper-based CRISPR detection, run an alternative reporter reaction following the incubation of the CRISPR reaction using a FAM-Biotin labeled DNA reporter. After combining the reagents into a 96-well plate, cover it with aluminum foil, then place the plate in an incubator at 37 degrees Celsius for 30 minutes.
Now add 80 microliters of PBS into a fresh well of a 96-well plate. Pipette 20 microliters of reporter reaction mixture into this well. Place a lateral flow paper strip to each well, and wait until the liquid reaches the top of the strip.
The chemically modified single-stranded DNA demonstrated Cas12a activation relative to the unmodified double stranded DNA and single stranded DNA. Modified DNA molecules in unprocessed urine showed colorimetric readout on lateral flow paper strips. The leading sample band indicated the release of detectable FAM through reporter cleavage triggered by urinary DNA.
