Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

The overall goal of this procedure is to harvest, concentrate, and preserve low abundance, low molecular weight biomarkers from biological fluids at appropriate concentrations. For standard protein assays, this is accomplished by first adding chemically functionalized hydrogel nanoparticles to the biological fluid, low molecular weight. Analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits.

The second step is to separate the particles from the biofluid by centrifugation. The nanoparticles concentrate the proteins of interest by several orders of magnitude. The nanoparticles are then washed with water to remove extraneous proteins.

The final step is to elute the captured proteins from the nanoparticles. Ultimately, western blotting, mass spectrometry, or amino assays are used to show the presence and or concentration of the previously undetectable harvested low abundance biomarkers. Generally, individuals new to this method will struggle because the nanoparticles need to be washed and resus suspended.

Without clogging the pipet tip, the nanoparticles do not resus suspend as easily as a cell P.So you must vigorously pipet up and down in order to adequately resus suspend the nanoparticles after the first incubation. With nanoparticles, it's important to rinse the walls of the tube in order to collect any nanoparticle residue that may have a adhere to the tube. To perform nanoparticle processing of serum samples dilute 500 microliters of human serum one to two with 50 millimolar tris, HCL pH seven in a micro centrifuge tube.

Add 500 microliters of poly and isop propyl acrylamide or poly nipam co monomers of acrylic acid core nanoparticles, and incubate for 15 minutes at room temperature. Then spin the sample at 16, 100 times G and 25 degrees Celsius for 10 minutes. In a centrifuge equipped with a fixed angle rotor, remove and discard the SUP natin before adding 500 microliters of sodium thiocyanate to the ate to the pellet, resuspend the nanoparticles by vigorously pipetting up and down multiple times.

Spin the sample as before and discard the supernatant before adding 500 microliters of milli Q water to the nanoparticle pellet. Once again, resus suspend the nanoparticles by vigorously pipetting up and down multiple times. After spinning the sample again, I had 300 microliters of fresh elucian, buffer and resuspend using the demonstrated technique.

Incubate for 15 minutes at room temperature. After a final spin of the sample, remove and save the EIT in a clean labeled micro centrifuge tube. Elute a second time and combine the two EITs into one micro centrifuge.Two.

Next, dry the EITs under nitrogen flow and a nitrogen evaporator manifold at 42.1 degrees Celsius with airflow set to six. Store the dried EIT at room temperature for overnight storage, or add negative 20 degrees Celsius for long-term storage prior to mass spectrometry Western blotting or EISA assays to perform nanoparticle processing of urine samples. Collect a minimum volume of 22 milliliters and store the specimens at negative 80 degrees Celsius until ready to analyze.

Thaw the frozen urine at room temperature or at four degrees Celsius overnight. Once thawed, mixed briefly on a vortex mixer. Then pour at least 22 milliliters of urine into a 50 milliliter conical bottom polypropylene tube.

Next, spin the urine in a centrifuge with a swing out rotor at 3, 700 times G for 15 minutes without disturbing the pellet. Decant the urine into a clean 50 milliliter conical bottom polypropylene tube and discard the pellet. Perform urinalysis using a multianalyte urine dipstick reagent strip.

Lay the reagent strip face up on a clean, dry paper towel. Use a disposable pipette to aspirate one milliliter of the urine. Quickly dispense one to two drops of urine on each test pad of the reagent strip.

Immediately start a timer that was previously set for two minutes at the time indicated on the reagent strip container. Record the qualitative results for the various analytes in the reagent strip by comparing the color of the individual reagent strips to the corresponding color coded indicators on the reagent container. Typical normal urine results are characterized by a pH of 5.5 to 7.0 and a specific gravity of 1.001 to 1.020.

The urine should be negative for blood protein, leukocytes, nitrite, glucose, ketone, bilirubin, and urobilinogen. Next, transfer 20 milliliters of the clarified urine into a clean 50 milliliter conical bottom polypropylene tube. Do not disturb any debris pellet that may be in the bottom of the urine tube from which you are removing the urine.

Add 200 microliters of nanoparticles to the 20 milliliter urine sample and mix briefly on a vortex mixer. Then incubate the urine nanoparticle mixture for 30 minutes at room temperature without rocking or mixing. Spin the urine nanoparticle suspension in a centrifuge equipped with a swing out rotor at 3, 700 times G for 10 minutes.

After removing and discarding the supernatant, add 500 microliters of Milli Q water to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Transfer the nanoparticle solution to a clean 1.5 milliliter micro centrifuge tube.

Next, spin the nanoparticles in a centrifuge equipped with a fixed angle rotor at 16, 100 times G for 10 minutes. Remove and discard the supernatant before repeating these steps with water. To wash the nanoparticles, add 20 microliters of fresh elution buffer to the nanoparticle pellet and resuspend the nanoparticles by vigorously pipetting up and down multiple times.

Incubate for 15 minutes at room temperature. Spin the nanoparticle samples again before removing the snat without disrupting the nanoparticle pellet and saving the supernatant in a clean 1.5 milliliter micro centrifuge tube. Discard the pellet in the biohazard trash.

Place the samples in a rack in a chemical fume hood. Open the caps and incubate at room temperature for 30 minutes. Alternatively, place the samples under nitrogen flow at 40 degrees Celsius until dry.

Add 15 microliters of tris glycine SDS sample buffer to the samples. Heat at 100 degrees Celsius in a dry heat block for five minutes with the caps open or until the volume left in the tube is no more than 20 microliters. Place the cap on the tubes and remove the tubes from the heat block.

The sample can be stored at negative 80 degrees Celsius or used immediately for downstream Western blot analysis. Silver stain 1D gel electrophoresis demonstrates nanoparticle capture and concentration of tumor necrosis factor alpha from human plasma. The band representing tumor necrosis factor alpha is only present in the nanoparticle EITs designated by the letter P for particles, but not in the supernatant nanoparticles Functionalized with two different chemical baits, acrylic acid or VSA core shell are able to harvest various concentrations of IL 17 from plasma western blotting of the supernatant and nanoparticle.

EIT clearly demonstrates the presence of IL 17 at the 17.5 kilodalton band in the eit, but not in the supernatants nanoparticle. Harvesting of urine samples can reveal bacterial antigens and cytokines in this silver stained SDS page. Analysis of urine samples samples in lanes U four and U six are nanoparticle EITs from urine samples containing RET ESAT six a mycobacterium tuberculosis associated protein, and IL two A cytokine.

These proteins are depicted by the prominent bands at 15 kilodaltons. After watching this video, you should have a good understanding of how gel nanoparticles can be used to harvest, concentrate, and preserve low abandoned biomarkers in biological fluids, while simultaneously excluding unwanted abandoned molecules, thus dramatically increasing the detection sensitivity of current analytical methods.