We thank the UCLA Biomedical Cyclotron Facility and Dr. Roger Slavik and Dr. Giuseppe Carlucci for generously providing [18F]fluoride for these studies and the UCLA NanoLab for support with equipment for chip fabrication.

CAUTION: This protocol involves the handling of radioactive materials. Experiments should not be undertaken without the necessary training and personal protective equipment and approval from the radiation safety office at your organization. Experiments should be performed behind radiation shielding, preferably in a ventilated hot cell
1. Fabrication of multi-reaction chips
NOTE: Batches of multi-reaction microdroplet chips are fabricated from 4&#34; silicon wafers using standard photolithography techniques, as previously decribed10 (Figure 1). This procedure will produce 7 chips each with 4 x 4 array of reaction sites.
<ol>
	<li>Place silicon wafer on the spin-coater chuck, ensuring that it is centered. Deposit 3 mL of polytetrafluoroethylene solution at the center of the wafer with a transfer pipette and coat wafer at a 1000 rpm for 30 s (500 rpm/s ramp).</li>
	<li>To solidify the coating, place the wafer on a 160 &#176;C hotplate for 10 min and then transfer to a 245 &#176;C hotplate for 10 min.</li>
	<li>Anneal the coating in a high-temperature oven at 340 &#176;C for 3.5 h under nitrogen atmosphere, followed by cooling to 70 &#176;C at a 10 &#176;C/min ramp.</li>
	<li>Place the silicon wafer on the spin-coater chuck, ensuring that it is centered. Pour 2 mL of positive photoresist at the center of the wafer using a transfer pipette, and then perform coating at 3000 rpm for 30 s (1000 rpm/s ramp).</li>
	<li>Solidify the photoresist by performing a soft bake of the wafer on a 115 &#176;C hotplate for 3 min.</li>
	<li>Install the wafer and photomask in a mask aligner and perform a 14 s exposure at 12 mW/cm2 lamp intensity and 356 nm wavelength in hard contact mode. This step uses a transparency mask containing the negative final polytetrafluoroethylene pattern, i.e., a 4&#34; diameter pattern of 4 copies of the 16-reaction chip, with reaction sites transparent and all other regions in opaque color.</li>
	<li>Submerge the wafer using 20 mL of photoresist developer solution in a glass container for 3 min with slight agitation to develop the exposed pattern.</li>
	<li>Rinse away the developing solution by submerging the wafer in a glass container with 20 mL of DI water for 3 min with slight agitation. Dry the wafer with a nitrogen gun.</li>
	<li>Remove the exposed polytetrafluoroethylene regions via reactive-ion etching (RIE) with oxygen plasma under the following conditions: 30 s exposure, 100 mTorr pressure, 200 W power, and 50 sccm oxygen flow.</li>
	<li>Dice the wafer into individual chips (7 total per wafer) using a silicon wafer cutter.</li>
	<li>Submerge each chip in acetone for 1 min to remove the photoresist, then isopropanol for 1 min. Finally, dry each chip with a nitrogen gun.</li>
	<li>Place dry chips in a glass container and cover with aluminum foil for storage until use.</li>
</ol>
2. Planning of the optimization study
NOTE: In this protocol, synthesis of the radiopharmaceutical [18F]fallypride is used as an example to illustrate high-throughput optimization (Figure 2). With a single chip, 16 simultaneous reactions can be performed, for example, with varied precursor concentration (8 different concentrations, n=2 replicates each). The conditions are mapped to reaction sites in Figure 3A. Adjustments can be made to this protocol to optimize other reaction parameters (e.g. reaction solvent, reaction volume, amount of TBAHCO3, etc.) or other radiopharmaceuticals.
<ol>
	<li>Select the reaction parameter(s) to be varied, the specific values to be used, and the number of replicates.</li>
	<li>Compute the number of chips needed to perform the experiment.</li>
	<li>For each chip, prepare a map of which reaction conditions will be used at each reaction site to assist with reagent preparation and performing the droplet reactions.</li>
</ol>
3. Preparation of reagents and materials for optimizing the radiosynthesis of [18F]fallypride
NOTE: The droplet-based radiosynthesis of [18F]fallypride (Figure 2) begins with the addition of [18F]fluoride and phase transfer catalyst (TBAHCO3) to the reaction site, followed by heating to evaporate water and leave a dried residue. Next, a droplet of precursor (tosyl-fallypride) in reaction solvent (thexyl alcohol and acetonitrile) is added and heated to perform the radiofluorination reaction. Finally, the crude product is collected from the chip for analysis. The reagent preparation and synthesis procedures should be adapted if performing optimization of a different tracer.
<ol>
	<li>Prepare a stock solution of the reaction solvent, consisting of thexyl alcohol and acetonitrile in a 1:1 by volume mixture. Ensure that the volume is enough to create the planned dilution series. In this example optimization, ~30 &#181;L is sufficient.</li>
	<li>Prepare a 30 &#181;L stock solution of precursor (tosyl-fallypride) in the reaction solvent with the maximum concentration to be explored (77 mM). Ensure that the volume is enough to perform the planned experiment. In this example optimization, ~30 &#181;L is sufficient.</li>
	<li>From the precursor stock solution and reaction solvent, perform 2x serial dilutions to prepare the different concentrations of the precursor solution. Ensure that the volume of each dilution is enough to perform the desired number of replicates for each condition. In this example optimization, ~15 &#181;L of each concentration is sufficient.</li>
	<li>Prepare microcentrifuge tubes to collect each crude reaction product using a permanent marker to label each tube with a unique number. Ensure that the total number of microcentrifuge tubes matches the number of conditions multiplied by the number of replicates (8 x 2 = 16).</li>
	<li>Prepare a stock of collection solution (10 mL) comprising 9:1 methanol:DI water (v/v). Aliquot 50 &#181;L into each of 16 additional labeled microcentrifuge tubes (one per reaction site on the chip).</li>
	<li>Prepare a [18F]fluoride stock solution in a 500 &#181;L microcentrifuge tube by mixing [18F]fluoride/[18O]H2O (~260 MBq [7 mCi]) with 75 mM TBAHCO3 solution (56 &#181;L) and diluting with DI water up to 140 &#181;L. 8 &#181;L of this solution will be loaded to each reaction site (containing ~15 MBq [0.40 mCi] of activity, and 240 nmol of TBAHCO3).</li>
</ol>
4. Parallel synthesis of [18F]fallypride with different precursor concentrations
NOTE: The chip is operated atop a heating platform (constructed as previously described13) consisting of a 25 mm x 25 mm ceramic heater, controlled using an on-off temperature controller using the internal thermocouple signal for feedback. Heater surface temperatures were calibrated using thermal imaging. If such a platform is not available, a pair of hot plates can be used (one at 105 &#176;C and one at 110 &#176;C).
<ol>
	<li>Load [18F]fluoride stock solution (with phase transfer catalyst).
	<ol>
		<li>Using a micropipette, load an 8 &#181;L droplet of [18F]fluoride stock solution on the first reaction spot of a multi-reaction chip. Measure the activity of the chip by placing it in a dose calibrator and record the time at which measurement is conducted.</li>
		<li>Remove the chip from dose calibrator and then load an 8 &#181;L droplet of [18F]fluoride stock solution on the second reaction spot. Measure the activity on the chip by placing it once again in the dose calibrator and record the time at which measurement is conducted.</li>
		<li>Repeat for all other reaction sites on the chip.</li>
		<li>Calculate the activity loaded per reaction spot by taking the activity measurement after loading the radioisotope and subtracting the previous measurement (decay-corrected) before that site was loaded.</li>
	</ol>
	</li>
	<li>Align the multi-reaction chip on the heater.
	<ol>
		<li>Add a thin layer of thermal paste on top of the ceramic heater.</li>
		<li>Carefully place the chip on top of the heater using tweezers to avoid the spill of the droplets, aligning the reference corner of the chip with the reference corner of the heater (as shown in Figure 3B). The chip will overhang the heater by a small amount.</li>
	</ol>
	</li>
	<li>Dry the [18F]fluoride and phase transfer catalyst.
	<ol>
		<li>Heat the chip for 1 min by setting the heater to 105 &#176;C in the control program to evaporate the droplets to dryness leaving a dried residue of [18F]fluoride and TBHACO3. After 1 min, cool the chip by turning off the heater and turning on the cooling fan with the control program.</li>
	</ol>
	</li>
	<li>Add the precursor solution.
	<ol>
		<li>Using a micropipette, add a 6 &#181;L solution of fallypride precursor on top of the dried residue on the first reaction site.</li>
		<li>Repeat for all other reaction sites on the chip. Use the optimization plan to determine which concentration of the dilution series is used for each reaction site.</li>
	</ol>
	</li>
	<li>Perform fluorination reaction.
	<ol>
		<li>Heat each chip to 110 &#176;C for 7 min using the control program to perform radiofluorination reaction. Afterwards, cool the chip by turning off the heater and turning on cooling fan with the control program.</li>
	</ol>
	</li>
	<li>Collect the crude products from the reaction sites.
	<ol>
		<li>Collect the crude product at the first reaction site by adding 10 &#181;L of collection solution from the designated microcentrifuge tube via micropipette. After waiting for 5 s, use the micropipette (with the same tip installed) to aspirate the diluted crude product and transfer to its corresponding labeled collection microcentrifuge tube.</li>
		<li>Repeat this process a total of 4 times using the same pipette tip for all operations.</li>
		<li>Repeat the collection process for all other reaction sites on the chip.</li>
	</ol>
	</li>
</ol>
5. Synthesis analysis to determine reaction performance and optimal conditions
<ol>
	<li>Determine the &#34;collection efficiency&#34; for the first reaction on the chip.
	<ol>
		<li>Place the microcentrifuge tube with the collected crude product of the first reaction spot in the dose calibrator to measure the activity. Record the measurement and time of the measurement.&#160;</li>
		<li>Calculate the collection efficiency by dividing the activity of the collected crude product by the starting activity measured for the same reaction site (decay-correcting the activity values to the same timepoint).</li>
		<li>Repeat for all other reaction sites on the chip.</li>
	</ol>
	</li>
	<li>Analyze the composition (fluorination efficiency) of each collected crude product. 
	NOTE: To make practical the analysis of all samples in a short time, fluorination efficiency is analyzed using a previously described high-throughput radio-thin layer chromatography (radio-TLC) approach14. This technique allows up to eight samples to be processed in parallel by spotting then side by side (5 mm pitch, 0.5 &#181;L per spot) on a single TLC plate, then developing together, and performing readout together using Cerenkov imaging14, 15. For the example optimization with 16 parallel reactions, 2 TLC plates are needed. Another option is to use radio- high-performance liquid chromatography (radio-HPLC) for analysis, though the time for separation, cleaning, and equilibration may limit the number of samples that can be analyzed.
	<ol>
		<li>For each TLC plate (50 mm x 60 mm), with a pencil, draw a line at 15 mm away from one 50 mm edge (bottom), and another line 50 mm away from the same edge. The first line is the origin line; the second is the solvent front line. Draw 8 small &#34;X&#34;s along the origin line at 5 mm spacing to define the sample spotting position for each of 8 &#34;lanes&#34;.</li>
		<li>Using a micropipette, transfer 0.5 &#181;L of the first crude product onto the TLC plate at the &#34;X&#34; for the first lane. Repeat for additional crude products (up to 8 per TLC plate). Wait for the crude product spots to dry on the TLC plate.</li>
		<li>For each TLC plate, develop using a mobile phase of 60% MeCN in 25 mM NH4HCO2 with 1% TEA (v/v) until the solvent front reaches the solvent front line. Wait for the solvent on the TLC plate to dry and then cover with a glass microscope slide (76.2 mm x 50.8 mm, 1 mm thick).</li>
		<li>Obtain a radioactivity image of each TLC plate by placing the plate in a Cerenkov imaging system for a 5 min exposure. Perform standard image corrections (dark current subtraction, flat field correction, median filtering, and background subtraction).</li>
		<li>Use region of interest (ROI) analysis for the first lane of the first TLC plate. Draw regions around each band visible in the lane. The software will compute the fraction of integrated intensity of each region (band) compared to the total integrated intensity of all regions (bands).</li>
		<li>With this mobile phase, the following bands are expected at the indicated retention factors: Rf = 0.0: Unreacted [18F]fluoride; Rf = 0.9: [18F]fallypride; Rf = 0.94: Side product. Determine the fluorination efficiency as the fraction of activity in the [18F]fallypride band.</li>
		<li>Repeat this analysis for all other lanes on all TLC plates. 
		&#8203;NOTE: If a Cerenkov imaging chamber is not available, a small animal (preclinical) in vivo optical imaging system can be used to image the TLC plates. Alternatively, a 2-dimensional TLC scanner can be used. Alternatively, if only a 1-dimensional TLC scanner is available, the TLC plates can be analyzed by cutting into strips with scissors (1 per lane), and scanning each strip individually.</li>
	</ol>
	</li>
	<li>Determine the crude radiochemical yield (crude RCY) for each reaction site.
	<ol>
		<li>Determine the crude RCY for the first crude product by multiplying the collection efficiency by the fluorination efficiency.</li>
		<li>Repeat for all other reaction sites.</li>
	</ol>
	</li>
	<li>Analyze the results
	<ol>
		<li>Aggregate values for any replicate experiments into an average and standard deviation.</li>
		<li>Plot the collection efficiency, fluorination efficiency, and crude RCY as a function of the parameter that was varied (precursor concentration in this example).</li>
		<li>Select the optimal conditions based on the desired criteria. Typically, this is the maximum crude RCY. Additionally, the point is often chosen in a region where the slope of the graph is relatively flat, indicating it is insensitive to small changes in the parameter, providing a more robust protocol.</li>
	</ol>
	</li>
</ol>

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The Regents of the University of California have licensed technology to Sofie, Inc. that was invented by Dr. van Dam, and have taken equity in Sofie, Inc. as part of the licensing transaction. Dr. van Dam is a founder and consultant of Sofie, Inc. The remaining authors declare no conflicts of interest. This work was supported in part by the National Cancer Institute (R33 240201).

Due to limitations of conventional radiochemistry systems that allow only one or a small number of reactions per day and consume a significant quantity of reagents per data point, only a tiny portion of the overall reaction parameter space can be explored in practice, and many times results are reported with no repeats (n=1). Compared to conventional systems, this multi-reaction droplet radiosynthesis platform makes it practical to accomplish more comprehensive and rigorous studies of radiosynthesis conditions while cons...

Positron-emission tomography (PET) radiopharmaceuticals are widely used as research tools to monitor specific in vivo biochemical processes and study diseases, and for the development of new drugs and therapies. Moreover, PET is a critical tool for diagnosing or staging disease and monitoring a patient&#39;s response to therapy1,2,3. Due to the short half-life of PET radioisotopes (e.g., 110 min for fluorine-18-labeled radiopharmaceuticals) and radiation hazard, these compounds are prepared using specialized automated systems operating behind radiation shielding and must be prepared just before use.
Current systems used to synthesize radiopharmaceuticals are designed to produce large batches that are divided up into many individual doses to share the production cost. While current systems are suitable for the production of widely used radiotracers like [18F]FDG (because multiple patient scans and research experiments can be scheduled in a single day), these systems can be wasteful for the production of novel radiotracers during early-stage development, or less commonly used radiotracers. Volumes that conventional systems use are typically in the 1-5 mL range, and the reactions require precursor amounts in the 1-10 mg range. Furthermore, using conventional radiosynthesizers is generally cumbersome during optimization studies since the apparatus becomes contaminated after use and the user must wait for radioactivity to decay before performing the next experiment. Aside from equipment cost, the cost of the radioisotope and reagents can, therefore, become very substantial for studies requiring production of multiple batches. This can occur, for example, during the optimization of synthesis protocols for novel radiotracers to achieve sufficient yield and reliability for initial in vivo imaging studies.
Microfluidic technologies have been increasingly used in radiochemistry to capitalize on several advantages over conventional systems4,5,6. Microfluidic platforms, including those based on 1-10 &#181;L reaction volumes7,8,9, have shown a significant reduction of reagent volumes and consumption of expensive precursors, as well as short reaction times. These reductions lead to lower costs, faster heating and evaporation steps, shorter and more straightforward downstream purification, an overall &#34;greener&#34; chemistry process10, and higher molar activity of the produced radiotracers11. These improvements make it more practical to perform more extensive optimization studies by lowering the reagent cost of each synthesis. Further benefits can be achieved by performing multiple experiments from a single batch of radioisotope in a single day. For example, microfluidic flow chemistry radiosynthesizers operating in &#34;discovery mode&#34; can sequentially perform dozens of reactions, each using only 10s of &#181;L reaction volume12.
Inspired by these advantages, a multi-reaction droplet array chip in which microvolume reactions are confined to an array of surface-tension traps on a silicon surface, created using a patterned Teflon coating, was developed. These chips enable multiple reactions at the 1-20 &#181;L scale to be performed simultaneously, opening the possibility to explore 10s of different reaction conditions per day, each with multiple replicates. In this paper, the utility of this new high-throughput approach for performing rapid and low-cost radiochemistry optimizations is demonstrated. Using multi-reaction droplet chips allows for convenient exploration of the impact of reagent concentrations and reaction solvent, and the use of multiple chips could enable the study of reaction temperature and time, all while consuming very low amounts of precursor.

<table><tbody><tr><td>2,3-dimethyl-2-butanol (thexyl alcohol)</td><td>Sigma-Aldrich</td><td>594-60-5</td><td>98%</td></tr><tr><td>Acetone</td><td>KMG Chemicals</td><td></td><td>Cleanroom LP grade</td></tr><tr><td>Ammonium formate (NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>540-69-2</td><td>97%</td></tr><tr><td>Anhydrous acetonitrile (MeCN)</td><td>Sigma-Aldrich</td><td>75-05-8</td><td>99.80%</td></tr><tr><td>Ceramic heater</td><td>Watlow</td><td>Utramic CER-1-01-0093</td><td>25 mm x 25 mm</td></tr><tr><td>Cerenkov imaging chamber</td><td>Custom built</td><td></td><td>Other instruments can be used for TLC plate readout including: small animal in vivo optical imaging system, 2D radio-TLC scanner, 1D radio-TLC scanner</td></tr><tr><td>DI water</td><td>Sigma-Aldrich</td><td>7732-18-5</td><td></td></tr><tr><td>Disposable transfer pipets, 3 mL</td><td>Falcon</td><td>13-680-50</td><td></td></tr><tr><td>Dose calibrator</td><td>Capintec, Inc.</td><td>CRC-25 PET</td><td></td></tr><tr><td>Fallypride</td><td>ABX Advanced Biochemical Compounds</td><td>1560.0010.000</td><td>Fallypride reference standard, &gt;95%</td></tr><tr><td>[&lt;sup&gt;18&lt;/sup&gt;F]fluoride in [&lt;sup&gt;18&lt;/sup&gt;O]H&lt;sub&gt;2&lt;/sub&gt;O</td><td>UCLA Ahmanson Biomedical Cyclotron Facility</td><td></td><td>Due to short half-life this must be obtained from local radiochemistry lab or commercial radiopharmacy</td></tr><tr><td>Glass cover plates (76.2 mm x 50.8 mm x 1 mm thick)</td><td>C&amp;amp;A Scientific</td><td>6101</td><td></td></tr><tr><td>Headway spin coater</td><td>Headway Research, Inc.</td><td>PWM50-PS-R790 Sipinner system</td><td>PWM50-control box, PS-motor, R790-bowl</td></tr><tr><td>High temperature oven</td><td>Carbolite</td><td>HTCR 6 28</td><td></td></tr><tr><td>Hot plate</td><td>Thermo Scientific</td><td>Super-Nuova HP133425</td><td></td></tr><tr><td>Isopropanol (IPA)</td><td>KMG Chemicals</td><td></td><td>Cleanroom LP grade</td></tr><tr><td>Mask aligner</td><td>Karl Suss</td><td>MA/BA6</td><td></td></tr><tr><td>Methanol (MeOH)</td><td>Sigma-Aldrich</td><td>67-56-1</td><td>&amp;ge;99.9%</td></tr><tr><td>Microcentrifuge tube</td><td>Eppendorf</td><td>0030 123.301</td><td>500 &amp;micro;L, colorless, polypropylene</td></tr><tr><td>Micropipette (0.5-10 &amp;micro;L)</td><td>Labnet</td><td>BioPette P3940-10</td><td></td></tr><tr><td>Micropipette (100-1000 &amp;micro;L)</td><td>Labnet</td><td>BioPette P3940-1000</td><td></td></tr><tr><td>Micropipette (10-100 &amp;micro;L)</td><td>Labnet</td><td>BioPette P3940-100</td><td></td></tr><tr><td>Micropipette tips (0.1-10 &amp;micro;L)</td><td>USA Scientific Inc Tips</td><td>11113810</td><td></td></tr><tr><td>Micropipette tips (2-200 &amp;micro;L)</td><td>BrandTech</td><td>13-889-143</td><td></td></tr><tr><td>Micropipette tips (50-1000 &amp;micro;L)</td><td>BrandTech</td><td>13-889-145</td><td></td></tr><tr><td>Photoresist developer solution</td><td>MicroChem</td><td>MEGAPOSIT MF-26A</td><td></td></tr><tr><td>Positive photoresist</td><td>MicroChem</td><td>MEGAPOSIT 220-7.0</td><td></td></tr><tr><td>Reactive-ion etcher (RIE)</td><td>Oxford Instruments</td><td>Plasma Lab 80 Plus</td><td></td></tr><tr><td>Silicon wafer cutter</td><td>Euro Tool</td><td>CSCB-431.00</td><td></td></tr><tr><td>Silicon wafer; 4&quot; diameter</td><td>Silicon Valley Microelectronics Inc.&amp;nbsp;</td><td>0017227-048</td><td>P type, boron doped, thickness 525 &amp;plusmn; 25 &amp;micro;m</td></tr><tr><td>Teflon AF 2400</td><td>Chemours&amp;nbsp;</td><td>D14896765</td><td>1% solids</td></tr><tr><td>Tetrabutylammonium bicarbonate (TBAHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>ABX Advanced Biochemical Compounds</td><td>808</td><td>Aqueous solution stabilized with ethanol, 0.075 M</td></tr><tr><td>Themal conducting paste</td><td>OMEGA</td><td>OT-201-2</td><td></td></tr><tr><td>TLC plates</td><td>Merck KGaA</td><td>1.05554.0001</td><td>Silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;, 50 mm x 60 mm, aluminum back</td></tr><tr><td>Tosyl-fallypride</td><td>ABX Advanced Biochemical Compounds</td><td>1550.004.000</td><td>Fallypride precursor, &gt;90%</td></tr><tr><td>Trimethylamine (TEA)</td><td>Sigma-Aldrich</td><td>75-50-3</td><td>&amp;ge; 99%</td></tr><tr><td>Tweezers</td><td>Cole-Parmer</td><td>UX-07387-08</td><td>Stainless steel, fine tip</td></tr></tbody></table>

optimization of radiochemical reactions using droplet arrays

Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction optimization or novel radiopharmaceutical development since each data point involves significant reagent consumption, and contamination of the apparatus requires time for radioactive decay before the next use. To address these limitations, a platform for performing arrays of miniature droplet-based reactions in parallel, each confined within a surface-tension trap on a patterned polytetrafluoroethylene-coated silicon "chip", was developed. These chips enable rapid and convenient studies of reaction parameters including reagent concentrations, reaction solvent, reaction temperature and time. This platform permits the completion of hundreds of reactions in a few days with minimal reagent consumption, instead of taking months using a conventional radiosynthesizer.

A representative experiment was performed to illustrate this method. Using 16 reactions, optimization studies of the radiopharmaceutical [18F]fallypride were performed by varying precursor concentration (77, 39, 19, 9.6, 4.8, 2.4, 1.2, and 0.6 mM) in thexyl alcohol:MeCN (1:1, v/v) as the reaction solvent. Reactions were performed at 110 &#176;C for 7 min. Collection efficiency, sample composition (i.e., proportions of [18F]fallypride product, unreacted [18...

Watch this Scientific Journal Video about Optimization of Radiochemical Reactions using Droplet Arrays at JoVE.com

Optimization of Radiochemical Reactions using Droplet Arrays

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction ...

optimization-of-radiochemical-reactions-using-droplet-arrays

Research

JoVE Journal

Chemistry

3.3K Views.  University of California Los Angeles (UCLA). This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

Video: Optimization of Radiochemical Reactions using Droplet Arrays

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Cancer Research

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

Engineering

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Bioengineering

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

Biology

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Efficient Synthesis of Carbon-11 Radiotracers Using the Loop Method Methylation

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...