Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Current clinical ovarian cancer detection measures are nonspecific and lack point of care detection of circulating tumor cells. Our photoacoustic flow system enables testing patient samples for specific ovarian cancer markers in the blood stream. This procedure provides a unique platform technology with improvements over the current techniques in terms of simplicity, low cost, promising limits of detection, and ability to be applied to broad applications.

Through this work, we aim to build off of our targeted nanoparticle system for ovarian cancer to test ex vivo patient samples for CTCs. This highly versatile PAFC system is able to expand for clinical use, testing the presence of a wide range of biomarkers in the blood. Proper optical alignment is critical for detection of targets within the photoacoustic flow cytometry system.

Furthermore, to ensure proper acoustic coupling, make sure there are no bubbles between the glass slide and transducer. Because of the custom nature of the flow system and its components, visualization of the method is beneficial for accurate reproduction. In a ventilated chemical fume hood, filter approximately 300 milliliters of deionized water through a sterile 0.2 micrometer filter.

Add 0.0134 grams of copper chloride and 100 milliliters of deionized water to a clean 250 milliliter round bottom flask to create a one millimolar copper chloride solution. Add 0.015 grams of folic acid to the flask and use a magnetic stir bar to mix the solution for approximately five minutes. Then mix 0.024 grams of sodium sulfide nonahydrate with 100 microliters of deionized water.

Use a 200 microliter pipette to slowly add this solution to the reaction mixture over a duration of approximately 10 seconds. Cap the reaction vessel. Place the vessel in an oil bath set to 90 degrees Celsius and continue stirring.

After approximately 15 minutes or when the oil bath has reached the temperature range between 85 and 90 degrees Celsius, allow the reaction to proceed for an additional hour. When the reaction is complete, remove the reaction vessel from the oil bath and let it cool at room temperature for 10 to 15 minutes before transferring it to an ice bath. Once the reaction is cooled, adjust the pH to approximately 10 using one molar sodium hydroxide.

Add the mixture to a 30 kilodalton centrifugation column in 15 milliliter batches and centrifuge at 3, 082 times g for 15 minutes to purify the reaction mixture. First, mix the folic acid capped copper sulfide nanoparticles at the proper concentration in fresh RPMI media. Add this nanoparticle solution to each well of the prepared 24-well plate that contains SK-OV-3 cells at a concentration of 400 micrograms per milliliter.

Incubate at 37 degrees Celsius with 5%carbon dioxide for two hours. After this, trypsinize the cells with 0.5 milliliters of 0.25%trypsin and EDTA. To neutralize the trypsin, add at least one milliliter of folic acid-free RPMI 1640 complete growth media and centrifuge the cells at 123 times g for six minutes.

To wash the cells, remove the supernatant, resuspend the cells in two milliliters of PBS and centrifuge at 123 times g for six minutes. Repeat this wash step twice to remove any unbound nanoparticles. Then resuspend the cells with one to two milliliters of a 2%Tween solution in PBS.

Count the cells using a hemocytometer and Trypan Blue. Dilute the cells in a solution of 2%Tween in PBS to the chosen concentration for detection. Use the provided three-dimensional STL file to 3D print the flow tank with either ABS thermoplastic or PLA plastic.

After printing the tank, clean and assemble the system for use. Place glass coverslips over the one millimeter by three millimeter slot and the one centimeter hole in the flow system and carefully seal with silicone to prevent leakage. Next, fit the capillary tube into the silicone-cured tubes.

Insert the tubes into the flow chamber through the side of the flow tank such that the glass capillary tube is directly above and in front of the three millimeter slot and the one centimeter hole. Seal the tubing with silicone. Then connect the transducer to an ultrasound pulser and receiver.

Amplify the signal with a 59 decibel gain. Connect the output of the filter to a multipurpose reconfigurable oscilloscope equipped with a built-in field programmable gate array. Connect one of the tubes coming from the flow chamber to a T junction that is connected to two syringe pumps at each branch.

Fill one of the syringe pumps with air and the other pump with the sample to be analyzed. Set the pump containing air to a flow rate of 40 microliters per minute and set the pump containing the sample to a flow rate of 20 microliters per minute. Next, connect the remaining tube exiting the flow system to a container of 10%bleach to dispose of cells after they exit the flow system.

Place the section of the quartz capillary tube in direct alignment with the transducer in the field view of the microscope to allow for careful placement of the optical fiber above the sample such that it illuminates the entire width of the tube. Irradiate the sample using an optical fiber channeling a diode-pumped solid-state laser operating at a wavelength of 1, 053 nanometers. Use a microscope mounted camera to record the flow, the firing of the laser, and the passage of samples through the flow system.

In this study, photoacoustic flow cytometry and the targeted copper sulfide targeting agent are used to detect ovarian circulating tumor cells. A typical TEM image of the synthesized nanoparticles show that the average size of a typical nanoparticle is approximately 8.6 nanometers. The horizontal and vertical diameters of each particle are then measured perpendicular to each other and further averaged.

The average hydrodynamic diameter for these particles is 73.6 nanometers. Copper sulfide nanoparticles have a characteristic absorbance curve which extends into the near infrared. There is a slight artifact around 850 nanometers that is caused by the switching of lasers by the spectrophotometer.

Fluorescence microscopy images of cells incubated with fluorescently tagged nanoparticles show that nanoparticle uptake can be visualized by the presence of fluorescence across the cell. Cells not incubated with nanoparticles show no fluorescent signal. The presence of this fluorescent signal indicates the successful uptake of the particles and their ability to be detected in the flow system.

Examples of typical data acquisition signals are shown here. The raw data indicates the differences in signal between the nanoparticle tagged cells, PBS, and folic acid capped copper sulfide nanoparticles. Utilizing custom lab view and MATLAB software, image reconstructions are made of the positive and negative controls in real time and post acquisition respectively.

Individual envelopers are subsequently converted into pixel values and displayed as independent columns. Clear differences in photoacoustic signal occur between PBS and the folic acid capped copper sulfide nanoparticles at a concentration of 100 micrograms per milliliter. During this procedure, it is critical to properly align the ultrasound transducer, microscope and fiber optic within the photoacoustic flow cytometry system.

Confirm system alignment by testing positive and negative controls. Due to the versatility of photoacoustic targeting techniques and the wide range of applications possible using PAFC, this technique paves the way for translationally important clinical and research applications. To ensure clinical application, further studies should focus on the testing of patient samples and the reduction of procedural steps.

The synthesis of the nanoparticles should take place in a chemical fume hood utilizing the proper protective equipment. Human cell lines must be handled according to your institution's guidelines. The use of the laser requires radiation safety training and appropriate PPE.

Laser usage and photoacoustic testing should only be performed by highly trained personnel.