We would like to thank David Rodriguez for assistance with the figure preparation and Precision Pathology Services (San Antonio, TX) for the use of its Navios EX flow cytometer.

All procedures need to be done using appropriate personal protective equipment.
1. Preparation of the TCPP stock solution, 1.0 mg/ mL
NOTE: This can be prepared monthly.
<ol>
	<li>Using an analytical balance, spatula, and weighing paper, weigh 49.0-50.9 mg of TCPP. Round the weight to 1/10 of a milligram. Set the measured amount of TCPP aside protected from light. 
	NOTE: Use a static gun if the weight reading is unstable.</li>
	<li>Determine the required amounts of purified water and isopropanol (IPA) from Table 1 based on the amount of TCPP weighed in step 1.1. Add the purified water and isopropanol to a 100 mL glass beaker, and cover with parafilm to protect from evaporation.</li>
	<li>Determine the required amount of sodium bicarbonate from Table 1 based on the amount of TCPP weighed in step 1.1.</li>
	<li>Using the analytical balance, spatula, and weighing paper, weigh the required amount of sodium bicarbonate determined in step 1.3. Round the weight to 1/10 of a milligram. 
	NOTE: Use a static gun if the weight reading is unstable.</li>
	<li>Add the sodium bicarbonate to the&#160;100 mL beaker containing purified water and isopropanol. Cover the solution with parafilm to protect from evaporation.</li>
	<li>Place the solution from step 1.5 on a stir plate, and stir until dissolved (approx. 10 min)</li>
	<li>Measure the pH to ensure that the sodium bicarbonate-containing solution from step 1.6 has a pH between 9 and 10.</li>
	<li>Slowly add the TCPP weighed in step 1.1 to the solution from step 1.7, and continue stirring until dissolved (~30 min). Protect from light during this step.</li>
	<li>Store in a glass or polypropylene container at room temperature and protected from light.</li>
</ol>
2. Preparation of 2-(N -morpholino)-ethanesulfonic acid (MES) and hemisodium salt buffer solution, 0.1 M, pH 6.0-6.2 (&#34;MES buffer&#34;)
NOTE: This must be prepared on the day of use and kept at room temperature.
<ol>
	<li>Weigh out 2.50 g of MES hemisodium salt, and add this to a 150 mL plastic bottle.</li>
	<li>Add 121 mL of purified water, and dissolve by manual shaking until no solid is visible.</li>
	<li>Measure the pH of the MES buffer to ensure that it is between 6 and 6.2.</li>
	<li>Maintain at room temperature for use on the same day.</li>
</ol>
3. N-(3-Dimethlyaminopropyl)-N&#39;-ethylcarbodiimide (EDC) powder
<ol>
	<li>Take the EDC powder out of the freezer, and let sit at room temperature until use in step 5.</li>
</ol>
4. Combining amine-functionalized polystyrene beads with TCPP solution
<ol>
	<li>Add 4.3 mL of the 0.1 M MES buffer solution prepared in step 2 to a 15 mL polypropylene tube.</li>
	<li>Vortex the amine-functionalized polystyrene bead suspension (10 &#181;m, 2.5% w/v) for 60 s at maximum speed.</li>
	<li>Add 288 &#181;L of this freshly vortexed bead suspension to the MES buffer from step 4.1.</li>
	<li>Vortex the MES/bead solution for 15 s at maximum speed.</li>
	<li>Vortex the 1 mg/mL TCPP solution prepared in step 1 for 60 s at maximum speed.</li>
	<li>Add 1.20 mL of this freshly vortexed TCPP stock solution to the MES/bead suspension from step 4.4.</li>
	<li>Vortex the MES/bead/TCPP suspension for 15 s at maximum speed.</li>
	<li>Cover the tube with foil while the EDC solution is prepared.</li>
</ol>
5. Preparation of N-(3-dimethlyaminopropyl)-N&#39;-ethylcarbodiimide (EDC) hydrocholoride (HCl) stock solution
NOTE: The EDC solution is perishable and should be used immediately following preparation.
<ol>
	<li>Add 20.0 mL of purified water to a new 50 mL conical tube.</li>
	<li>Weigh out 200 mg of EDC HCl from step 3, and add it to the water (step 5.1).</li>
	<li>Vortex the EDC HCL for 15 s at maximum speed to generate a clear solution.</li>
</ol>
6. Preparation of the EDC HCl/MES working solution
NOTE: The EDC HCl/MES solution is perishable and should be used immediately following preparation.
<ol>
	<li>Add 54.0 mL of MES buffer solution (prepared in step 2) to a 150 mL plastic bottle.</li>
	<li>Add 6.0 mL of the EDC HCl stock solution (prepared in step 5) to the MES buffer solution, and mix by shaking for 10 s.</li>
</ol>
7. Labeling the beads with TCPP
<ol>
	<li>Add 4.5 mL of EDC working solution (from step 6) to the 15 mL polypropylene tube containing the beads and TCPP in MES buffer (step 4.7).</li>
	<li>Place the tube in an inverting rotator at 35 rpm for 16 h at room temperature and protected from light.</li>
	<li>Centrifuge the tube at room temperature for 10 min at 1,000 &#215; g.</li>
	<li>Aspirate the supernatant, and resuspend the beads in 0.8 mL of Hanks&#39; balanced salt solution (HBSS).</li>
	<li>Transfer the bead solution to an amber polypropylene vial with a 1 mL pipette, and store at 4 &#176;C until further use. 
	&#8203;NOTE: The beads are stable for at least 3 months at this point.</li>
</ol>
8. Quality check (QC) of the TCPP beads by flow cytometry
NOTE: The QC should be centered on whether the median fluorescence intensity (MFI) of the TCPP beads is sufficiently bright for their intended use and the amount of particulates generated by the procedure. See the representative results section for more details.
<ol>
	<li>Label one 5 mL polystyrene tube as &#34;TCPP negative beads.&#34; 
	NOTE: The negative beads are different from the amine-functionalized beads used for labeling. See the Table of Materials.</li>
	<li>Label another tube as &#34;TCPP positive beads.&#34;</li>
	<li>Label another tube as &#34;Rainbow beads.&#34;</li>
	<li>Aliquot 300 &#181;L of ice-cold HBSS into the tubes labeled with &#34;TCPP negative beads&#34; and &#34;TCPP positive beads.&#34;</li>
	<li>Add 500 &#181;L of ice-cold HBSS into the tube labeled as &#34;Rainbow beads.&#34;</li>
	<li>Vortex the non-functionalized polystyrene (unlabeled) bead suspension briefly at maximum speed (2-3 s), and add 10 &#181;L of it to the tube labeled &#34;TCPP negative beads.&#34;</li>
	<li>Vortex the TCPP-labeled bead suspension (finalized in step 7.5) briefly at maximum speed, and add 3 &#181;L of it to the &#34;TCPP positive beads&#34; tube.</li>
	<li>Vortex the Rainbow bead suspension briefly at maximum speed, and add two drops of it into the &#34;Rainbow beads&#34; tube.</li>
	<li>Keep all tubes on ice, covered, and protected from light.</li>
	<li>Initiate the appropriate daily startup procedures of the flow cytometer, and perform a QC to verify the optimal fluids and laser alignment. 
	NOTE: For this part of the protocol, it is assumed that the operator is trained in the use of the flow cytometer that is available, including the procedures of standardizing the light scatter and fluorescence intensity, as well as the basic principles of calculating the correct compensation matrix.</li>
	<li>Run the Rainbow beads and the TCPP beads without changing the voltage settings in between the different runs.
	<ol>
		<li>Run and collect 10,000 events of the Rainbow beads.</li>
		<li>Perform a rinse with water, and collect 10,000 events of the TCPP-negative beads.</li>
		<li>Perform a rinse with water, and collect 10,000 events of the TCPP-positive beads.</li>
		<li>Perform a 1 min water rinse. 
		NOTE: It is important to perform a rinse with water after running the TCPP beads. If the TCPP is not rinsed from the lines in the cytometer, there is the possibility that residual TCPP can label cells in the next tube to be acquired.</li>
		<li>Perform the appropriate cleaning and shutdown protocols specific to the manufacturer&#39;s instructions for the cytometer. 
		&#8203;NOTE: For representative results, see Figure 1.</li>
	</ol>
	</li>
</ol>
9. Bead filtration
NOTE: If the QC of the beads by flow cytometry (step 8) shows a high proportion of particulates (70% or higher), consider filtering the bead suspension using the protocol below (Figure 2).
<ol>
	<li>Add 3.20 mL of ice-cold HBSS to 0.8 mL of the TCPP bead suspension finalized in step 7.5 (creating a five-fold dilution).</li>
	<li>Vortex the diluted bead suspension at maximum speed for 15 s.</li>
	<li>Remove the plunger from a disposable 5 mL syringe.</li>
	<li>Fit the syringe with a glass fiber tip filter (5 &#181;m, 13 mm diameter).</li>
	<li>Add 4 mL of HBSS to the syringe.</li>
	<li>Add 0.5 mL of the vortexed diluted bead suspension (step 9.2).</li>
	<li>Use the plunger to filter the suspension through the syringe/filter setup at approximately 2 drops/s.</li>
	<li>Wash the beads by drawing 5 mL of fresh HBSS into the syringe through the filter at approximately 2 drops/s.</li>
	<li>Push the HBSS out again into the waste container at approximately 2 drops/s.</li>
	<li>To remove the beads from the filter, draw another 5 mL of fresh HBSS into the syringe through the filter.</li>
	<li>Carefully remove the filter from the syringe.</li>
	<li>Eject the bead suspension from the syringe into a 50 mL conical centrifuge tube.</li>
	<li>Place the filter back on the syringe, and repeat steps 9.10-9.12 four more times. Then&#160;discard the filter and syringe.</li>
	<li>Repeat steps 9.2-9.13 until all the beads from step 9.1 have been filtered. Use a fresh syringe and filter each time.</li>
	<li>Centrifuge the filtered bead suspensions for 10 min at 1,000 &#215; g at room temperature.</li>
	<li>Aspirate the supernatant of each 50 mL tube, and gently resuspend the beads, combining them in 0.5 mL of fresh HBSS.</li>
	<li>Transfer the beads with a p1,000 micropipette to a new amber, glass, or polypropylene vial, and store at 4 &#176;C.</li>
	<li>Repeat step 8 to determine if the proportion of TCPP-related particulates in the filtered bead suspension has decreased. For representative results, see Figure 3.</li>
</ol>

Porphyrin-Modified Beads in Flow Cytometry

<ol>
	<li>Josefsen, L. B., Boyle, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+diagnostic+and+therapeutic+roles+of+porphyrins+and+phthalocyanines+in+photodynamic+therapy,+imaging+and+theranostics.">Unique diagnostic and therapeutic roles of porphyrins and phthalocyanines in photodynamic therapy, imaging and theranostics.</a> Theranostics. 2 (9), 916-966 (2012).</li><li>Tsolekile, N., Nelana, S., Oluwafemi, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrin+as+diagnostic+and+therapeutic+agent.">Porphyrin as diagnostic and therapeutic agent.</a> Molecules. 24 (14), 2669 (2019).</li><li>Gunaydin, G., Gedik, M. E., Ayan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+the+treatment+and+diagnosis+of+cancer-A+review+of+the+current+clinical+status.">Photodynamic therapy for the treatment and diagnosis of cancer-A review of the current clinical status.</a> Frontiers in Chemistry. 9, 686303 (2021).</li><li>Berg, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrin-related+photosensitizers+for+cancer+imaging+and+therapeutic+applications.">Porphyrin-related photosensitizers for cancer imaging and therapeutic applications.</a> Journal of Microscopy. 218, 133-147 (2005).</li><li>Kessel, D., Reiners, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-activated+pharmaceuticals:+Mechanisms+and+detection).">Light-activated pharmaceuticals: Mechanisms and detection).</a> Israel Journal of Chemistry. 52 (8-9), 674-680 (2012).</li><li>Didamson, O. C., Abrahamse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+photodynamic+diagnosis+and+therapy+for+esophageal+cancer:+Potential+role+of+functionalized+nanomedicine.">Targeted photodynamic diagnosis and therapy for esophageal cancer: Potential role of functionalized nanomedicine.</a> Pharmaceutics. 13 (11), 1943 (2021).</li><li>Harada, Y., Murayama, Y., Takamatsu, T., Otsuji, E., Tanaka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Aminolevulinic+acid-induced+protoporphyrin+IX+fluorescence+imaging+for+tumor+detection:+Recent+advances+and+challenges.">5-Aminolevulinic acid-induced protoporphyrin IX fluorescence imaging for tumor detection: Recent advances and challenges.</a> International Journal of Molecular Sciences. 23 (12), 6478 (2022).</li><li>Bochenek, K., Aebisher, D., Mi&#281;dzybrodzka, A., Cie&#347;lar, G., Kawczyk-Krupka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+bladder+cancer+diagnosis+-+The+role+of+autofluorescence+and+photodynamic+diagnosis.">Methods for bladder cancer diagnosis - The role of autofluorescence and photodynamic diagnosis.</a> Photodiagnosis and Photodynamic Therapy. 27, 141-148 (2019).</li><li>Iwaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry-based+photodynamic+diagnosis+with+5-aminolevulinic+acid+for+the+detection+of+minimal+residual+disease+in+multiple+myeloma.">Flow cytometry-based photodynamic diagnosis with 5-aminolevulinic acid for the detection of minimal residual disease in multiple myeloma.</a> The Tohoku Journal of Experimental Medicine. 249 (1), 19-28 (2019).</li><li>Patriquin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+lung+cancer+with+meso+tetra+(4-carboxyphenyl)+porphyrin-labeled+sputum.">Early detection of lung cancer with meso tetra (4-carboxyphenyl) porphyrin-labeled sputum.</a> Journal of Thoracic Oncology. 10 (9), 1311-1318 (2015).</li><li>Pan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+introduction+to+porphyrin+compounds+used+in+tumor+imaging+and+therapies.">A brief introduction to porphyrin compounds used in tumor imaging and therapies.</a> Mini Reviews in Medicinal Chemistry. 21 (11), 1303-1313 (2021).</li><li>Nishida, K., Tojo, T., Kondo, T., Yuasa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+correlation+between+porphyrin+accumulation+in+cancer+cells+and+functional+positions+for+application+as+a+drug+carrier.">Evaluation of the correlation between porphyrin accumulation in cancer cells and functional positions for application as a drug carrier.</a> Scientific Reports. 11 (1), 2046 (2021).</li><li>Lin, Y., Zhou, T., Bai, R., Xie, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+approaches+for+the+enhancement+of+porphyrin+skeleton-based+photodynamic+therapy.">Chemical approaches for the enhancement of porphyrin skeleton-based photodynamic therapy.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 35 (1), 1080-1099 (2020).</li><li>Kou, J., Dou, D., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrin+photosensitizers+in+photodynamic+therapy+and+its+applications.">Porphyrin photosensitizers in photodynamic therapy and its applications.</a> Oncotarget. 8 (46), 81591-81603 (2017).</li><li>Wezgowiec, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-assisted+delivery+of+photofrin+to+human+breast+carcinoma+cells.">Electric field-assisted delivery of photofrin to human breast carcinoma cells.</a> The Journal of Membrane Biology. 246 (10), 725-735 (2013).</li><li>Palasuberniam, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+kinase+inhibitors+enhance+aminolevulinic+acid-mediated+protoporphyrin+IX+fluorescence+and+PDT+response+in+triple+negative+breast+cancer+cell+lines.">Small molecule kinase inhibitors enhance aminolevulinic acid-mediated protoporphyrin IX fluorescence and PDT response in triple negative breast cancer cell lines.</a> Journal of Biomedical Optics. 26 (9), 098002 (2021).</li><li>Kim, B., Bohandy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopy+of+porphyrins.">Spectroscopy of porphyrins.</a> Johns Hopkins APL Technical Digest. 2 (3), 153-163 (1981).</li><li>Uttamlal, M., Sheila Holmes-Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+excitation+wavelength+dependent+fluorescence+of+porphyrins.">The excitation wavelength dependent fluorescence of porphyrins.</a> Chemical Physics Letters. 454 (4), 223-228 (2008).</li><li>Zhang, Y. Z., Kemper, C., Bakke, A., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+flow+cytometry+compensation+standards:+internally+stained+fluorescent+microspheres+with+matched+emission+spectra+and+long-term+stability.">Novel flow cytometry compensation standards: internally stained fluorescent microspheres with matched emission spectra and long-term stability.</a> Cytometry. 33 (2), 244-248 (1998).</li><li>Monard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+spectral+cytometry+toolbox:+Coupling+fluorescent+proteins+and+antibodies+to+microspheres.">Building a spectral cytometry toolbox: Coupling fluorescent proteins and antibodies to microspheres.</a> Cytometry. Part A. 101 (10), 846-855 (2022).</li><li>Byrd, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polystyrene+microspheres+enable+10-color+compensation+for+immunophenotyping+of+primary+human+leukocytes.">Polystyrene microspheres enable 10-color compensation for immunophenotyping of primary human leukocytes.</a> Cytometry. Part A. 87 (11), 1038-1046 (2015).</li><li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensation+in+flow+cytometry.">Compensation in flow cytometry.</a> Current Protocols in Cytometry. , (2002).</li><li>Kabe, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrin+accumulation+in+mitochondria+is+mediated+by+2-oxoglutarate+carrier.">Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier.</a> The Journal of Biological Chemistry. 281 (42), 31729-31735 (2006).</li><li>Bederka, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sputum+analysis+by+flow+cytometry;+An+effective+platform+to+analyze+the+lung+environment.">Sputum analysis by flow cytometry; An effective platform to analyze the lung environment.</a> PLoS One. 17 (8), e0272069 (2022).</li><li>. US6838248B2 - Compositions and methods for detecting pre-cancerous conditions in cell and tissue samples using 5, 10, 15, 20-tetrakis (carboxyphenyl) porphine Available from: <a target="_blank" href="https://patents.google.com/patent/US68248B2/en?oq=US+patent+6838248+B2">https://patents.google.com/patent/US68248B2/en?oq=US+patent+6838248+B2</a> (2005)</li><li>. Method of using 5,10,15,20-tetrakis(carboxyphenyl)porphine for detecting cancers of the lung Available from: <a target="_blank" href="https://www.osti.gov/deopatents/biblio/7117152">https://www.osti.gov/deopatents/biblio/7117152</a> (1992)</li><li>Grayson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality-controlled+sputum+analysis+by+flow+cytometry.">Quality-controlled sputum analysis by flow cytometry.</a> Journal of Visualized Experiments. (174), e62785 (2021).</li><li>Anjali, K., Christopher, J., Sakthivel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruthenium-based+macromolecules+as+potential+catalysts+in+homogeneous+and+heterogeneous+phases+for+the+utilization+of+carbon+dioxide.">Ruthenium-based macromolecules as potential catalysts in homogeneous and heterogeneous phases for the utilization of carbon dioxide.</a> ACS Omega. 4 (8), 13454-13464 (2019).</li><li>Yadav, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+preparation+and+applications+of+organo-functionalized+porous+materials.">Recent advances in the preparation and applications of organo-functionalized porous materials.</a> Chemistry. 15 (17), 2588-2621 (2020).</li><li>. US7670799B2 - Method for making 5,10,15,10-tetrakis (carboxyphenyl) porphine (TCPP) solutions and composition compromising TCPP Available from: <a target="_blank" href="https://patents.google.com/patent/US7670799B2/en">https://patents.google.com/patent/US7670799B2/en</a> (2023)</li><li>Shimizu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+affinity+beads+for+identifying+drug+receptors.">High-performance affinity beads for identifying drug receptors.</a> Nature Biotechnology. 18 (8), 877-881 (2000).</li><li>Anderson, G. W., Zimmerman, J. E., Callahan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+esters+of+N-hydroxysuccinimide+in+peptide+synthesis.">The use of esters of N-hydroxysuccinimide in peptide synthesis.</a> Journal of the American Chemical Society. 86 (9), 1839-1842 (1964).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. , (2013).</li><li>El-Faham, A., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+coupling+reagents,+more+than+a+letter+soup.">Peptide coupling reagents, more than a letter soup.</a> Chemical Reviews. 111 (11), 6557-6602 (2011).</li><li>Hulspas, R., O'Gorman, M. R. G., Wood, B. L., Gratama, J. W., Sutherland, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+the+control+of+background+fluorescence+in+clinical+flow+cytometry.">Considerations for the control of background fluorescence in clinical flow cytometry.</a> Cytometry Part B. 76 (6), 355-364 (2009).</li><li>Hoffman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization,+calibration,+and+control+in+flow+cytometry.">Standardization, calibration, and control in flow cytometry.</a> Current Protocols in Cytometry. , (2005).</li><li>Ethirajan, M., Chen, Y., Joshi, P., Pandey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+porphyrin+chemistry+in+tumor+imaging+and+photodynamic+therapy.">The role of porphyrin chemistry in tumor imaging and photodynamic therapy.</a> Chemical Society Reviews. 40 (1), 340-362 (2011).</li><li>Beharry, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+photodynamic+therapy:+New+probes+for+cancer+imaging+and+treatment.">Next-generation photodynamic therapy: New probes for cancer imaging and treatment.</a> Biochemistry. 57 (2), 173-174 (2018).</li><li>El-Far, M., Pimstone, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+28+porphyrins+and+their+abilities+to+localize+in+mammary+mouse+carcinoma:+Uroporphyrin+I+superior+to+hematoporphyrin+derivative.">A comparative study of 28 porphyrins and their abilities to localize in mammary mouse carcinoma: Uroporphyrin I superior to hematoporphyrin derivative.</a> Progress in Clinical and Biological Research. 170, 661-672 (1984).</li></ol>

All authors are employees of bioAffinity Technologies.

Despite the many applications of porphyrins in cancer diagnosis and therapeutics2, there is limited literature on their potential use as a flow cytometric reagent for the identification of cancerous versus non-cancerous cell populations in primary human tissues24,25,26. Our research on the flow cytometric analysis of human sputum24,27 requires the...

Porphyrins have been of interest for many years in the biomedical field owing to their fluorescence and tumor-targeting properties1,2,3. Therapeutic applications such as photodynamic therapy (PDT) and sonodynamic therapy (SDT) entail the systemic administration of a porphyrin to a cancer patient, the accumulation of the drug in the tumor, and the localized exposure of the tumor to a laser light of a specific wavelength or ultrasound. The exposure to laser light or ultrasound leads to the generation of reactive oxygen species by the porphyrin and subsequent cell death4,5. In photodynamic diagnosis (PDD), porphyrin fluorescence is used to distinguish cancer cells from normal cells6. In this context, protoporphyrin IX, a natural fluorescent porphyrin that accumulates in tumors upon the systemic or local injection of its precursor, 5-aminolevulinic acid (5-ALA), is used to identify gastrointestinal stromal tumors, bladder cancer, and brain cancer7,8. More recently, 5-ALA treatment was explored as an approach to detect minimal residual disease in multiple myeloma9. Our laboratory has been using the tetraaryl porphyrin TCPP (5,10,15,20-tetrakis-(4-carboxyphenyl)-21,23H-porphine) for its ability to selectively stain lung cancer cells and cancer-associated cells in human sputum samples, which is a property that has been exploited in slide-based and flow cytometric diagnostic assays10.
Some porphyrins are bifunctional in that they can be used as therapeutic and diagnostic agents2,11. In biomedical research, such bifunctional porphyrins are used to evaluate how their ability to selectively target and kill cancer cells is a function of their structure as well as how it is affected by the presence of other compounds12,13,14,15,16. Both the cellular uptake of porphyrins and their cytotoxicity can be measured on a flow cytometric platform in a high-throughput manner. The absorption and emission spectra of fluorescent porphyrins are complex, but most flow cytometric platforms are equipped to correctly identify them. The absorption spectrum of fluorescent porphyrins is characterized by a strong absorption band in the 380-500 nm range, known as the Soret band. Two to four weaker absorption bands are generally observed in the 500-750 nm range (Q bands)17. A blue&#160;488 nm laser, present in most flow cytometers, or a violet laser (405 nm) can generate light of the appropriate wavelength to excite porphyrins. The emission spectra of porphyrins typically display peaks in the 600-800 nm range18, which results in very little spectral overlap with fluorescein isothiocyanate or phycoerythrin (PE) fluorophores but considerable overlap with other often-used fluorophores, such as allophycocyanin (APC), as well as tandem fluorophores, such as PE-Cy5 and others. Therefore, when using porphyrins in multi-color flow cytometry assays, single-fluorophore controls are essential to adequately correct the spillover of fluorescence in channels other than the one designated to measure the porphyrin&#39;s fluorescence.
Ideally, the single-fluorophore controls used to calculate the spillover matrix for a panel of fluorophores (also called &#34;compensation controls&#34;) should consist of the same cell type(s) as the sample. However, using the sample for this purpose is not optimal if there is very little sample to begin with or if the target population within the sample is very small&#160;(for example, if one wants to look at minimal residual disease or cancer cells at the early stages of the disease). A useful alternative to cells is beads coupled with the same fluorophore that is used to analyze the sample. Many such beads are commercially available; these beads are either prelabeled with the desired fluorophore (prelabeled fluorophore-specific beads)19,20, or a fluorescently labeled antibody can be attached to them (antibody capture beads)20,21. While commercial compensation beads are available for many fluorophores, such beads are unavailable for porphyrins, despite their increasing use in basic and clinical research.
In addition to sample preservation and appropriately sized positive versus negative populations, the other advantages of using beads as compensation controls are the ease of preparation, low background fluorescence, and excellent stability over time22. The potential disadvantage of using beads as a compensation control is that the emission spectrum of the fluorescent antibody captured on beads may differ from that of the same antibody used to label the cells. This may be of specific importance when using a spectral flow cytometer20. Therefore, the development of beads as a compensation control needs to be performed on the flow cytometer that will be used for the assay for which the beads are developed. Moreover, the development of the beads needs to include a comparison with cells labeled with the same fluorescent staining reagent.
Here, we describe the preparation of TCPP amine-functionalized polystyrene compensation beads, whose median fluorescence intensity in the detection channel was comparable to that of TCPP-labeled cells in sputum, and their use as compensation controls for flow cytometry. The autofluorescence of equivalent, non-functionalized beads was sufficiently low for their use as negative fluorescence compensation controls. In addition, these beads demonstrated stability in storage for nearly 1 year.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amber plastic vials, 2 mL, U- bottom, polypropylene</td>
			<td>Research Products International&#160;&#160;</td>
			<td>ZC1028-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine-funtionalized polystyrene divinylbenzene crosslinked (PS/DVB) beads, 10.6 &#956;m diameter, 2.5% w/v aqueous suspension, 3.82 x 107 beads/mL, 7.11 x 1011 amine groups/ bead</td>
			<td>Spherotech</td>
			<td>APX-100-10</td>
			<td>Diameter spec. 8.0-12.9 um, suspension 2.5% w/v 3.82 x 107 beads/mL, 7.11 x 1011 amine groups/ bead</td>
		</tr>
		<tr>
			<td>Conical tubes, 50 mL, Falcon</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>with appropriate rotor</td>
		</tr>
		<tr>
			<td>Disposable polystyrene bottle with cap, 150 mL</td>
			<td>Fisher Scientific</td>
			<td>09-761-140</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC (N- (3- dimethylaminopropyl)- N&#39;- ethylcarbodiimide hydrochloride), &#8805;98%</td>
			<td>Sigma</td>
			<td>03450-1G</td>
			<td>CAS No:&#160; 25952-53-8</td>
		</tr>
		<tr>
			<td>FlowJo Single Cell Analysis Software (v10.6.1)</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips, 22 x 22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-540-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass fiber syringe filters (Finneran, 5 &#181;m, 13 mm diameter)</td>
			<td>Thomas Scientific</td>
			<td>1190M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides, 275 x 75 x 1 mm</td>
			<td>Fisher Scientific</td>
			<td>12-550-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Fisher Scientific</td>
			<td>14-175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, ACS grade</td>
			<td>Fisher Scientific</td>
			<td>AC423830010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical pipette, 1 channel, 100-1000 uL with tips</td>
			<td>Eppendorf</td>
			<td>3123000918</td>
			<td></td>
		</tr>
		<tr>
			<td>MES (22- (N- mopholino)- N&#39;- ethanesulfonic acid, hemisodium salt</td>
			<td>Sigma</td>
			<td>M0164</td>
			<td>CAS No:&#160; 117961-21-4</td>
		</tr>
		<tr>
			<td>Navios EX flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX-40 microscope with DP73 camera and 40X objective with cellSens software</td>
			<td>Olympus</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Pasteur pipettes, glass, 5.75&#34;</td>
			<td>Fisher Scientific</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter (UB 10 Ultra Basic)</td>
			<td>Denver Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette controller (Drummond)</td>
			<td>Pipete.com</td>
			<td>DP101</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Syringe, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>14955452</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Particles (non-functionalized), SPHERO,&#160; 2.5% w/v, 8.0-12.9 &#181;m</td>
			<td>Spherotech</td>
			<td>PP-100-10&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes, 15mL, conical</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene tubes, round bottom&#160;</td>
			<td>Fisher Scientific</td>
			<td>14-959-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainbow Beads (Spherotech URCP-50-2K)</td>
			<td>Fisher Scientific</td>
			<td>NC9207381</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes, disposable - 10 mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes, disposable - 25 mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-576</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>CAS No:&#160; 144-55-8</td>
		</tr>
		<tr>
			<td>TCPP (meso-tetra(4-carboxyphenyl)porphine)&#160; Frontier Scientific&#160;</td>
			<td>Fisher Scientific</td>
			<td>50-393-68</td>
			<td>CAS No:&#160; 14609-54-2</td>
		</tr>
		<tr>
			<td>Tecan Spark Plate Reader (or similar)</td>
			<td>Tecan Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube revolver/rotator</td>
			<td>Thermo Fisher</td>
			<td>88881001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
	</tbody>
</table>

porphyrin-modified beads for use as compensation controls in flow cytometry

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. 
Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

This protocol for the TCPP labeling of beads is relatively fast and efficient. Figure 1 shows a representative outcome of the TCPP bead-labeling process as determined by flow cytometry. Figure 1A shows the standardized profile of Rainbow beads, as detected in the appropriate channel for detecting TCPP. These beads serve as a QC for the standardization of the laser voltages for the detection of TCPP by the flow cytometer. Figure 1B s...

Watch this Scientific Journal Video about Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry at JoVE.com

Author Spotlight: Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry  

The protocol describes how porphyrin-based compensation beads for flow cytometry are prepared by the reaction of amine-functionalized polystyrene beads with the porphyrin TCPP and the amide coupling reagent EDC. A filtration procedure is used to reduce the particulate byproducts.

Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or ...

porphyrin-modified-beads-for-use-as-compensation-controls-flow

Research

JoVE Journal

Chemistry

2.0K Views.  bioAffinity Technologies. The protocol describes how porphyrin-based compensation beads for flow cytometry are prepared by the reaction of amine-functionalized polystyrene beads with the porphyrin TCPP and the amide coupling reagent EDC. A filtration procedure is used to reduce the particulate byproducts. 

Video: Author Spotlight: Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry  

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment allows for testing of various proposed ocean / hydrothermal fluid chemistries that could have driven reactions toward the origin of life in environments on the early Earth, early Mars, or even other worlds such as the icy moons of the outer planets. We show experiments that include growth of chemical garden structures under anoxic conditions simulating the early Earth, inclusion of trace components of phosphates / organics in the injection solution to incorporate them into the structure, a switch of the injection solution to introduce a secondary precipitating anion, and the measurement of membrane potentials generated by chemical gardens. Using this method, self-assembling chemical garden structures were formed that mimic the natural chimneys precipitated at submarine hydrothermal springs, and these precipitates can be used successfully as flow-through reactors by feeding through multiple successive &#8220;hydrothermal&#8221; injections.

We describe chemical garden formation via injection experiments that allow for laboratory simulations of natural chemical garden systems that form at submarine hydrothermal vents.

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment ...

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

HKUST-1 as a Heterogeneous Catalyst for the Synthesis of Vanillin

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately 200 different odorant compounds in addition to vanillin. The natural extraction of vanillin (from the orchid Vanilla planifolia, Vanilla tahitiensis and Vanilla pompon) represents only 1% of the worldwide production and since this process is expensive and very long, the rest of the production of vanillin is synthesized. Many biotechnological approaches can be used for the synthesis of vanillin from lignin, phenolic stilbenes, isoeugenol, eugenol, guaicol, etc., with the disadvantage of harming the environment since these processes use strong oxidizing agents and toxic solvents. Thus, eco-friendly alternatives on the production of vanillin are very desirable and thus, under current investigation. Porous coordination polymers (PCPs) are a new class of highly crystalline materials that recently have been used for catalysis. HKUST-1 (Cu3(BTC)2(H2O)3, BTC = 1,3,5-benzene-tricarboxylate) is a very well known PCP which has been extensively studied as a heterogeneous catalyst. Here, we report a synthetic strategy for the production of vanillin by the oxidation of trans-ferulic acid using HKUST-1 as a catalyst.

The conversion of trans-ferulic acid to vanillin was achieved by heterogeneous catalysis. HKUST-1 was employed in this synthesis and the essential step in the catalytic process was the generation of unsaturated metal sites. Thus, when the catalyst was activated under vacuum, full vanillin conversion (yield of 95%) was obtained.

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately ...

1,3,5-Triphenylbenzene and Corannulene as Electron Receptors for Lithium Solvated Electron Solutions

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic hydrocarbons (PAH), namely 1,3,5-triphenylbenzene and corannulene, as electron receptors. The solid PAHs were first dissolved in tetrahydrofuran (THF) to form a solution. Metallic lithium was then dissolved into these PAH/THF solutions to yield either blue or greenish blue solutions, colors which are indicative of the presence of solvated electrons. Conductivity measurements at ambient temperature carried out on 1,3,5-triphenylbenzene-based LiSES, denoted by LixTPB(THF)24.7 (x = 1, 2, 3, 4), showed an increase of conductivity with increase of Li:PAH ratio from x = 1 to 2. However, the conductivity gradually decreased upon further increasing the ratio. Indeed the conductivity of LixTPB(THF)24.7 for x = 4 is even lower than for x = 1. Such behavior is similar to that of the previously reported LiSES prepared from biphenyl and naphthalene. Conductivity versus temperature measurements on corannulene-based LiSES, denoted by LixCor(THF)247 (x = 1, 2, 3, 4, 5), showed linear relationships with negative slopes, indicating a metallic behavior similar to biphenyl and naphthalene-based LiSES.

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using 1,3,5-triphenylbenzene (TPB) and corannulene as electron receptors.

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...