Cite this: Lab Chip, 2013, 13, 2328

Radiochemistry on chip: towards dose-on-demandsynthesis of PET radiopharmaceuticals3

Received 10th January 2013,Accepted 25th March 2013

DOI: 10.1039/c3lc00055a

www.rsc.org/loc

Valentina Arima,*a Giancarlo Pascali,bc Oliver Lade,d Hans R. Kretschmer,d

Ingo Bernsdorf,d Victoria Hammond,e Paul Watts,ef Francesco De Leonardis,e MarkD. Tarn,e Nicole Pamme,e Benjamin Z. Cvetkovic,g Petra S. Dittrich,g Nikola Vasovic,h

Russell Duane,h Aleksandar Jaksic,h Antonella Zacheo,ai Alessandra Zizzari,a

Lucia Marra,a Elisabetta Perrone,a Piero A. Salvadorib and Rosaria Rinaldiai

We have developed an integrated microfluidic platform for producing 2-[18F]-fluoro-2-deoxy-D-glucose

(18F-FDG) in continuous flow from a single bolus of radioactive isotope solution, with constant product

yields achieved throughout the operation that were comparable to those reported for commercially

available vessel-based synthesisers (40–80%). The system would allow researchers to obtain radio-

pharmaceuticals in a dose-on-demand setting within a few minutes. The flexible architecture of the

platform, based on a modular design, can potentially be applied to the synthesis of other radiotracers that

require a two-step synthetic approach, and may be adaptable to more complex synthetic routes by

implementing additional modules. It can therefore be employed for standard synthesis protocols as well as

for research and development of new radiopharmaceuticals.

Introduction

Positron emission tomography (PET) is a powerful molecularimaging technique largely used for diagnosis and therapycontrol.1 It is based on the injection into a patient of a drugwith a known biological activity labelled with a positron-emitting nuclide. In vivo imaging of the resulting radioactivityallows monitoring of several biological pathways to assess thehealth of the patient undergoing the exam.

Currently, the most widespread tracer for PET is 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG), thanks to its well-knownbiological activity and the relatively long half-life of 18F-fluoride (109.7 min). 18F-FDG is synthesized in quantitiessufficient for the formulation of multiple doses from a singleproduction run (the batch approach), followed by shipmentfrom the production/cyclotron site (the so-called ‘‘satellitecentre’’ philosophy), usually within a 4 h delivery time, door-to-door to PET centres. The production of 18F-FDG via thisbatch approach has several drawbacks and is associated withhigh cost and time losses. A more convenient approach wouldbe to produce a single dose of tracer when needed for a singlePET scan and then have the possibility to promptly restart theproduction line if another dose is needed (the dose-on-demand approach, known as DOD).2,3 Additionally, the DODapproach may be extended to produce different tracers fromthe same batch of starting radioisotope, thus reducingpersonnel exposure while allowing maximum flexibility torespond to the user’s schedule.

Currently, DOD production of 18F-FDG is not achievablewith the traditional vessel-based techniques that are inwidespread use throughout PET centres. Microfluidics andflow chemistry represent innovative technologies that arecompatible with DOD requirements, and which have thepotential to increase radiochemical productivity in terms ofhigher yields, shorter reaction times, reduced precursorconsumption, lower environmental impact and flexible experi-mental planning.4,5

aNational Nanotechnology Laboratory (NNL)-Institute of Nanoscience (NANO), CNR,

Via per Arnesano 16, 73100 Lecce, Italy. E-mail: valentina.arima@nano.cnr.it;

Fax: +39 0832 298230; Tel: +39 0832 298218bInstitute of Clinical Physiology, CNR, Via Moruzzi 1, 56124 Pisa, ItalycAustralian Nuclear Sciences and Technology Organization, Life Sciences Division,

Locked Bag 2001, Kirrawee DC, NSW 2232, AustraliadSiemens AG, Siemensdamm 50, 13629 Berlin, GermanyeDepartment of Chemistry, University of Hull, Cottingham Road, Hull, HU6 7RX, UKfInnoVenton: NMMU Institute for Chemical Technology, Nelson Mandela

Metropolitan University, PO Box 77000, Port Elizabeth 6031, RSAgDepartment of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Str.

10, CH-8093 Zurich, SwitzerlandhTyndall National Institute, Lee Maltings, Dyke Parade, Cork, IrelandiUniversita del Salento, Dipartimento di Matematica e Fisica ‘‘E. De Giorgi’’, ex

Collegio Fiorini Campus extraurbano, via per Arnesano, 73100 Lecce, Italy

3 Electronic supplementary information (ESI) available: Information andphotographs of the Labtrix system employed for the synthesis, test data for thesolvent exchange module and radioactivity detector, a description of the heaterfoils and alignment device, and a detailed description of the operation of theROC platform for 18F-FDG synthesis. See DOI: 10.1039/c3lc00055a

Lab on a Chip

PAPER

2328 | Lab Chip, 2013, 13, 2328–2336 This journal is � The Royal Society of Chemistry 2013

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article OnlineView Journal | View Issue

The validity of the microfluidic approach to perform fastand high yield radiolabelling reactions has been demonstratedin several pioneering works.6–8 Automated microfluidicdevices have also been demonstrated for the synthesis of 18F-FDG in quantities sufficient for mouse imaging.9,10 The firsthuman use of a PET radiotracer produced by a batch-reactormicrofluidic device was recently reported.11

Additionally, the microfluidic approach has been imple-mented in a new generation of commercial apparatuses,available from Advion, Future Chemistry and Scintomics.These systems have demonstrated the flexibility and effective-ness of this approach for radiochemical synthesis, but noneare readily applicable to the typical heavy-duty cycles ofproduction centres.

In terms of dedicated miniaturised systems, Elizarov et al.12

developed a very compact coin-shaped microreactor thatallows for the production of injectable 18F-FDG by entirelyprocessing a single bolus of starting radioisotope solution.However, the system was fabricated in polydimethylsiloxane(PDMS), a material degradable by radioactive solutions,13 andwas fully dedicated to 18F-FDG synthesis, hence it was notapplicable to repeated, quasi-continuous production of severalDOD radiotracers. Here, to move beyond the state-of-the-artand towards a meaningful DOD approach, we proposed amicrofluidic approach to radiochemistry based on the designand assembly of a new modular system (named hereafter the‘‘radiochemistry-on-chip’’, or ‘‘ROC’’, platform), and thedevelopment of a simplified synthetic route for 18F-FDGproduction.

Conventional 18F-FDG synthesis is a complex process. 18F-fluoride is produced in aqueous solution via a cyclotron, andincorporated into a pharmaceutical compound, typically bynucleophilic substitution under suitable activation conditions.The amount of 18F-fluoride produced by the cyclotron is verylow, hence a pre-concentration step is required. Activation isgenerally achieved by drying the aqueous solution, replacing itwith an organic aprotic solvent, and adding alkali or earth-alkali salts with or without the use of organic cryptands. Theprocedure most commonly employed for 18F-fluoride incor-poration utilises the acetonitrile/H2O azeotrope for drying of18F-fluoride in the presence of potassium carbonate (K2CO3)

and Kryptofix 2.2.2 (K222), the latter being a cryptand that trapsthe potassium ions, leaving the 18F-fluoride ions free to react.The nucleophilicity of this 18F-fluoride is large enough that,even in an organic phase such as acetonitrile, it is able to reactvia SN2 nucleophilic substitution with precursor compoundsthat feature suitable leaving groups. Unfortunately, the use ofa precursor amenable to such nucleophilic displacement(hence bearing leaving groups such as tosylates, halogens,nitro groups etc.) is essential for the labelling reaction, whichmeans that it is sometimes necessary to use a ‘‘pre-precursor’’that, once labelled with high radiochemical yield in the firststep, has to be further (and quickly) chemically modified inorder to obtain the final, desired product. Therefore, the mainoperations required to synthesise 18F-FDG, as shown in Fig. 1,are:

N Step 1) Pre-concentration of the aqueous 18F-fluoridespecies from irradiated 18O-water.

N Step 2) Solvent exchange from water to acetonitrile(CH3CN), usually via azeotropic distillation.

N Step 3) Labelling reaction of mannose triflate 1 with 18F-fluoride to form acetylated-18F-FDG 2.

N Step 4) Solvent exchange from acetonitrile to water.N Step 5) Hydrolysis reaction of acetylated-18F-FDG 2 to 18F-

FDG 3 using sodium hydroxide, though alternatively hydro-chloric acid can be used.

N Step 6) Purification of the crude 18F-FDG mixture by solid-phase extraction (SPE).

We have developed a modular system, the ROC platform, inwhich these steps can be performed within individualmicrofluidic modules, with the intention that these modulescan be swapped in or out when fresh chips are required, andthat the production of radiotracers other than 18F-FDG couldbe produced simply by changing the appropriate modules. Inthis first trial of the system, microfluidic devices weredeveloped that performed each of the synthetic processes for18F-FDG production (Fig. 2a–b).

Module 1 consisted of a chamber packed with anionexchange resin in order to perform the pre-concentration (step1) and first solvent exchange from water to acetonitrile (step 2)operations.14 Module 2 featured a serpentine channel designfor the radiolabelling reaction of mannose triflate to

Fig. 1 Typical synthetic process for the production of 18F-FDG 3, involving the radiolabelling of mannose triflate 1 with 18F-fluoride to form acetylated-18F-FDG 2,which is then hydrolysed to give the final product 3.

This journal is � The Royal Society of Chemistry 2013 Lab Chip, 2013, 13, 2328–2336 | 2329

Lab on a Chip Paper

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online

acetylated-18F-FDG (step 3). Module 3 was a solvent exchangedevice designed to transfer acetylated-18F-FDG from acetoni-trile to water (step 4).15 Module 4 was a second serpentinemicroreactor for the hydrolysis of acetylated-18F-FDG to 18F-FDG (step 5).

These modules were then integrated within the platformitself in order to perform the synthesis of 18F-FDG on theassembled instrument (Fig. 2c). Purification of the crude 18F-FDG (step 6) was implemented off-platform via a series ofmicrofluidic modules connected to the outlet of module 4.16

Experimental

Reagents for chemical synthesis

All chemical reagents were purchased from Sigma-Aldrich orABX (Radeberg, Germany) and used without further purifica-tion, unless otherwise stated. All resins were obtained fromABX (Germany). 18F-fluoride was produced by a PETtrace

cyclotron (GE Healthcare, USA) using a silver 1.5 mL target and13 MeV irradiation at 30 mA for variable times (5–60 min).

Reagents and substrates for chip fabrication

Borosilicate substrates covered with chromium film werepurchased from Telic (Valencia, CA, USA). Hydrofluoric acid(48%), hydrochloric acid (36%), and ammonium fluoride(40%) were purchased from Sigma-Aldrich. Photoresist(AZ9260) and photodeveloper (AZ400K) were purchased fromNowel s.r.l. (Baranzate, Milan, Italy), and chromium etchantfrom Poletto Aldo s.r.l. (Noventa di Piave, Venice, Italy).Photomasks were designed using CleWin software, andprinted by J. D. Photo-tools Ltd. (Oldham, Lancashire, UK).

Design and fabrication of the microfluidic devices

Microfluidic devices were fabricated in borosilicate glass withoverall dimensions of 2.5 cm 6 4.7 cm (Fig. 2b). Thefabrication processes of modules 1 and 3 are described inref. 14 and 15, respectively. Briefly, module 1 contained a 30mm long 6 4.7 mm wide 6 300 mm deep chamber that couldbe packed with particles for performing the pre-concentrationof 18F-fluoride. The design featured a 50 mm deep triangularweir near a single outlet hole (368 mm diameter) that allowedtrapping of the particles, while a single inlet hole (1 mmdiameter) enabled the introduction of particles and solutions.This design was also used to prepare solid-phase extractionchips for purification of the synthesised 18F-FDG, althoughthese were not integrated directly into the final ROC apparatusbut instead added on to the end of the synthetic process.16

Module 3 consisted of two etched plates bonded together: abase plate containing 10 mm wide and 5 mm deep capillariesfor the separation of the liquid and gas phases, and a top platefeaturing input channels and a separation channel that were400 mm wide and 100 mm deep. Inlet and outlet holes were 1/1699 in diameter. Modules 2 and 4 were T-serpentine geometrymicroreactors with microchannel lengths of 900 mm and 270mm, respectively, and internal volumes of 10 and 4 mL.Microchannels were 200 mm wide for module 2 and 250 mmwide for module 4, and were etched to a depth of 50 mm inboth microreactors. Two inlet holes and one outlet hole, eachof 1/1699 diameter, allowed the introduction of reagents andthe collection of product, respectively.

Fabrication of the microfluidic devices was performed usingstandard photolithography and wet etching procedures.17

Briefly, the chip design pattern was transferred from thephotomask to the photoresist layer of the borosilicate glass viaUV light exposure. The photoresist was then developed,followed by removal of the exposed chromium layer, thusrevealing the channel design onto the glass substrate itself.The channels were etched into the glass using a solution ofbuffered oxide etchant (BOE, a mixture of 6 : 1 hydrofluoricacid (48%) to ammonium fluoride (40%)) with the addition of20% v/v hydrochloric acid (36%), at a constant temperature of70 uC. After etching, access holes were drilled into the plateusing diamond-coated drill bits to allow introduction andremoval of fluids. Next, rigorous cleaning of the etchedsubstrate and a glass top plate was performed using piranhasolution (3 : 1 sulphuric acid (98%) to hydrogen peroxide (30%

Fig. 2 (a) Schematics showing the principle of operation of the four modules:(1) pre-concentration module, (2) first microreactor for the radiolabelling ofmannose triflate 1 with 18F-fluoride, (3) solvent exchange module, and (4) thesecond microreactor for the hydrolysis of acetylated-18F-FDG 2 with NaOH. (b)Photographs of the individual glass modules. (c) Photograph of the fourmodules integrated within the ROC platform.

2330 | Lab Chip, 2013, 13, 2328–2336 This journal is � The Royal Society of Chemistry 2013

Paper Lab on a Chip

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online

v/v)), after which the two plates were washed with water,aligned, and thermally bonded in a furnace at 620 uC for 3 h.

Pre-concentration device (module 1)

The operation and performance of the 18F-fluoride pre-concentration module has been demonstrated previously,albeit for the synthesis of 18F-fluoroethyltosylate rather than18F-FDG.14 In brief, the microfluidic chamber was packed withanion exchange resin (PS-HCO3 particles, 100 mm diameter),then flushed with 2 mL ethanol and 2 mL water at 1000 mLmin21 for activation prior to its insertion into the ROCplatform.

Optimisation of FDG synthetic steps via commercialmicrofluidic apparatus (towards modules 2 and 4)

Microreactor studies were carried out using the Labtrix Startsystem from Chemtrix BV (Netherlands), as this offered thedesired temperature range (25–180 uC) for performing reac-tions, and featured a variable back pressure regulator (set to 15bar) so that solvent would remain liquid at these temperatures.The temperature control system for the setup was a MelcorCorporation thermoelectric temperature controller (MTTC-1410) (Fig. S1a in the ESI3), while the syringe pump suppliedas part of the system was a Chemyx Fusion 200. The glassmicroreactor (Fig. S1b, ESI3) supplied with the instrument wascontained within a holder that could be placed over the heatedelement. The internal volume of the serpentine microreactors(Fig. S2–S3, ESI3) was 10 mL, and residence times of reagentswithin the microchannels were calculated according to thetime spent by this volume over the heated element, with flowrates adjusted accordingly. Mannose triflate 1 dissolved inacetonitrile (0.015 M) and a solution of fluoride (1.5equivalents) were pumped through a reactor at fixed residencetimes in order to investigate the effect of temperature on thereaction. In the ‘‘cold’’ (non-radioactive) reactions performedin anhydrous solvent, the percentage conversion was deter-mined by HPLC (LC-10AD, Shimadzu) with an electrochemicaldetector (Coulochem II, ESA (now part of Thermo Scientific)).A calibration graph for both mannose triflate 1 and FDG 3 wasplotted in order to determine the percentage conversion andthe mass balance in the reaction. The conversions in the ‘‘hot’’(radioactive) labelling reactions were determined via radio-TLC detection, using a 95 : 5 acetonitrile to water mobilephase and a silica stationary phase. Results were confirmed byradio-HPLC, using a Phenomenex Synergi Hydro-RP columnwith a 43 : 57 acetonitrile : water mobile phase, at a flow rateof 1 mL min21.

Solvent exchange device (module 3)

The microchip module for solvent exchange consisted of threefunctional sections (Fig. 2, and ref. 15). Firstly, a liquidreaction mixture of acetonitrile and water was introduced intoone inlet, while nitrogen gas was introduced into a separateinlet, creating an annular flow of the liquid with the gas in thecore. Secondly, a serpentine channel was used to allowevaporation of the more volatile solvent (here acetonitrile)into the gas stream. In the final section, the liquid wasseparated from the solvent-enriched gas stream. This wasaccomplished by two series of side capillaries, through which

the aqueous liquid was withdrawn and collected, while the gasstream remained within the main channel. The generaloperation and performance of the solvent exchange modulewas evaluated for various mixtures of acetonitrile and water,and for a range of flow rates and nitrogen.15

Additionally, the module was tested by employing a solutionof K222/K2CO3/18F-fluoride dissolved in an acetonitrile–watermixture. It was observed that the radioactivity remained in theliquid phase during the evaporation process, while the coldcondensed vapours exiting the gas outlet did not carry anysubstantial radioactivity. Due to the low detection limit forpositron emitting compounds, the use of 18F-fluoride enabledvery sensitive monitoring of this separation process. Fig. S4(ESI3) shows the detected radioactivity in the liquid phase(blue line) and in the condensed gas phase (red line) from theoutlets during the experiment. Less than 1% of the injectedradioactive liquid exited from the gas outlet, underlining thehigh separation efficiency of the device under the appliedconditions (20 mL min21 liquid flow rate, 0.7 bar nitrogenpressure, and a temperature of 55 uC).

Purification modules

The fabrication and operation of the purification modules aredescribed elsewhere.16 In brief, these chips were based on thepre-concentration chips (module 1), except that four moduleswere connected in a ‘‘train’’ configuration for performingsolid-phase extraction (SPE) of the unwanted components inthe crude 18F-FDG mixture. The chambers, in order, were filledwith: (i) cation exchange resin (PS–H+, 100 mm diameter), (ii)anion exchange resin (PS–HCO3 particles, 100 mm diameter),(iii) normal phase resin (ALOX N, 60–100 mm diameter), and(iv) reversed phase resin (HR-P, 50–100 mm diameter). Thepurification modules were connected to the outlet of thehydrolysis module such that the crude 18F-FDG was fed intothe train of SPE chips.

Radiation detection modules

Miniature radiation probes were developed to monitorchanges in radioactivity in different parts of the microfluidicsystem. The probes were based on novel SiliconPhotoMultiplier technology coupled with LSO (cerium-dopedlutetium oxyorthosilicate) scintillators, developed by SensL(www.sensl.com, Cork, Ireland). Silicon PhotoMultiplier tech-nology offers advantages over legacy photomultiplier tubes interms of size, ruggedness, operating voltage, and cost. Thedetector was tested prior to its use in the ROC platform, whereit showed excellent linearity with 18F-FDG activity (see Fig. S5,ESI3).

Electronic and mechanical components of the ROC platform

Components utilised in the ROC platform were purchasedfrom the following sources: medium mass flow meters fromBronkhorst Mattig (Karmen, Germany); neMESYS high preci-sion low pressure syringe pump from Cetoni (Korbußen,Germany); two adjustable DC power supplies (10 V to 16.5 V)with an maximum output current of 3 A from Seltec (nowPheonix Contact, Blomberg, Germany); data acquisition device(PCI 6254) and connector block (SCB-68) from NationalInstruments (Munich, Germany) for sensor signal acquisition,

This journal is � The Royal Society of Chemistry 2013 Lab Chip, 2013, 13, 2328–2336 | 2331

Lab on a Chip Paper

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online

heater and valve control; one PC from Siemens with aLabVIEW application for automation of the ROC platform;pressure sensors (PAA21Y) from Omega Newport(Deckenpfronn, Germany); switching valves (MXX777-601)from Rheodyne (IDEX Health & Science, Wertheim-Mondfeld); heating elements (FHK-TBA) and heaterstat tem-perature controller (CT 198, DC controller) from Minco(Saarbrucken, Germany); adjustable back-pressure regulatorvalves (300 psi) from Upchurch (IDEX Health & Science, OakHarbor, USA); shut-off valves for medium pressure applica-tions from Techlab (Braunschweig, Germany); small, highpressure rotary valves and selector, two types of PEEK tubing(0.25 mm OD and 0.18 mm ID) and 1/1699 fittings connectingmass flow meters and pressure sensors together from Omnifit(Cambridge, UK).

Design of the ROC platform control system

The system consisted of three main assemblies: the fluidicplatform (FP, see Fig. S8a, ESI3), the pumping platform (PP, seeFig. S8c–d, ESI3), and the control platform (CP) (see Fig. S8b,ESI3). The fluidic platform was located inside a standard hotcell as this was where the synthesis of 18F-FDG was performed,while the pumping platform and control platform werepositioned outside, with cables and tubing directed into thehot cell to the fluidic platform.

The fluidic platform (Fig. 3) featured a two-level stage thatconsisted of: (i) Four plug-in modules situated on the lowerlevel of the stage. The modules (1–4) were positioned side-by-side, and were fitted into PEEK (polyether ether ketone) chipholders that were operated via a drawer-type system to allowquick and easy insertion/removal of the glass chips. Analignment device was fabricated to assist in the accurateplacement of the glass modules with heater foils and tubing(Fig. S7, ESI3). Although only four modules were present in theROC platform in these experiments, there was space for afurther four modules that could include product purificationchips or dedicated modules for producing different radio-pharmaceuticals. Steel contacting bridges were fabricated thatheld the chips within the PEEK holders, and also supportedthe PEEK tubing, Omnifit fittings, and O-rings in order toguarantee leak-free manipulation of fluids within the micro-fluidic devices. The contacting bridges used for the micro-reactor chips and the solvent exchange chip additionally heldelectrical contacting pins that were used for actuatingintegrated Minco heaters (Ni/Fe foils that could be operatedup to 225 uC) (Fig. S6, ESI3); (ii) A 2-position, 6-port switchingvalve (the Auto Valve Switch) that allowed switching betweenthe two operating modes of the system (‘‘production’’ and ‘‘no-production’’). The system variables were controlled by threemass flow meters, one pressure regulator for nitrogen gas, twoadjustable back-pressure regulator valves, and four pressuresensors mounted on the upper level of the stage. All PEEKtubing present in the system was connected to the relevantdevices via Omnifit fittings; (iii) Four absolute pressuresensors with a range up to 10 bar mounted on the platform,and two types of flow sensor that monitored three syringe-driven flows; (iv) Standard thermocouples to measure thetemperature of the glass chip surfaces.

The pumping platform was equipped with: (i) A CetonineMESYS high precision, low pressure syringe pump forpulsation free dosage in the nano- and milliliter ranges inorder to feed the fluidic platform with FDG reagents andsolvents. The pump was positioned vertically in front of a steelwell for bubble-free filling of five glass syringes; (ii) The fiveglass syringes were connected by tubing to five manual rotaryvalves that were placed on top of the steel well plate. Oneadditional manual rotary valve was connected directly to aswitching valve situated on the fluidic platform; (iii) Severalglass bottle stations at the rear of the steel well plate, fromwhich the glass syringes were filled with different solvents andreagents; (iv) Five one-way check valves between the fluidicplatform and the pumping platform to avoid any backflow ofthe radioactive solutions through the tubing. Details on thechip adjustment device, operation, and high temperatureprocessing for performing 18F-FDG synthesis are reported inthe ESI.3

The control platform was composed of a computer running aLabView program to drive the neMESYS pumping station, andto manage the voltage supply unit that enabled pressuremonitoring of all four modules and the temperature control ofmodules 2–4.

Fig. 3 (a) CAD schematic, and (b) photograph of the overall ROC platform,showing the locations of the microfluidic modules (lower shelf) and the valvingand pressure regulator system (upper shelf).

2332 | Lab Chip, 2013, 13, 2328–2336 This journal is � The Royal Society of Chemistry 2013

Paper Lab on a Chip

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online

18F-FDG synthesis in the ROC platform

Kryptofix 2.2.2 (30 mg) was dissolved in 4 mL of acetonitrilewith 160 mL aqueous potassium carbonate (5% w/v) added,and this solution was charged into syringe 1 (Fig. S9, ESI3).Mannose triflate 1 (20 mg) was dissolved in acetonitrile (4 mL)and charged into syringe 2. Syringe 3 was loaded with 0.1 MNaOH. The irradiated 18O-water collected from the cyclotron(1.5 mL) was delivered into the pre-concentration module at500 mL min21 using an external syringe pump while the ROCapparatus was set to the ‘‘no-production’’ modality. Uponturning the Auto Switch Valve to ‘‘production’’ modality,syringe 1 and 2 started to deliver the solutions at 10 mL min21,while syringe 3 delivered NaOH at 100 mL min21. After 10 minof continuous operation, 100 mL fractions were collected andanalyzed by radio-TLC for up to 120 min. Upon completion ofthe delivery, a manual valve was turned towards syringe 4,containing pure acetonitrile, in order to rinse the system withinert solvent. Radiation detection was achieved using thesilicon photomultipliers developed for this project, andconfirmed with PIN photodiodes supplied with an AdvionNanoTek system that was present in the lab.

Results and discussion

The operations of modules 1 (pre-concentration) and 3(solvent exchange) have already been described in detailelsewhere.14,15 Following their individual testing and optimi-sation the modules were integrated into the ROC platform asdescribed here. Parallel to the development of these devices,studies were undertaken to determine the most convenientmicrofluidic-based strategy for performing the synthetic stepsof 18F-FDG production, i.e. the radiolabelling reaction (step 3)and the hydrolysis reaction (step 5).

Optimisation of synthetic steps

Prior to integration into the ROC platform, optimisation of theon-chip synthetic steps was achieved by employing ‘‘cold’’chemistry conditions in order that experiments could beperformed without the use of radioactivity or hot cells, thusaffording added flexibility to the investigation. To this end,‘‘cold’’ potassium fluoride solution was employed as thefluoride source, as an analogue to ‘‘hot’’ 18F-fluoride producedby a cyclotron. Syntheses were performed on a commerciallyavailable microreactor system (Labtrix Start), which was usedto identify the optimal conditions for the conversion ofmannose triflate 1 to acetylated-FDG 2, and from 2 to FDG 3,with the two steps involved initially being optimised sepa-rately. The microreactor employed for the radiolabellingreaction (step 3) is illustrated in Fig. S2 (ESI3), with thestarting reagents of 1 and KF/K222/K2CO3, each dissolved inanhydrous acetonitrile, delivered via the inlets into the 10 mLmicroreactor. The applied flow rates were varied to study thecorrelation of different residence times over the heatedelement with fluorination yield.

Fig. 4a shows the results for the fluorination optimisation of1 to 2 at three different residence times (15, 30 and 60 s). It wasnoticed that for each residence time, the conversion increased

with increasing temperature up to 150 uC, after which the yielddecreased due to decomposition of both 1 and 2. In addition,the conversion was higher using longer residence times, withthe optimum conditions determined to be a 60 s residencetime and a temperature in the range of 125–150 uC. The ratioof fluoride to 1 was also investigated, since in ‘‘hot’’ chemistrythe mannose triflate 1 is present in vast excess. The results ofthis set of reactions is reported in Fig. 4b, showing that theincrease of the amount of 1 has a beneficial effect on theconversion to 2 (reported in terms of percentage with regard tostarting concentration of fluoride, to be comparable to ‘‘hot’’chemistry).

This set of studies was subsequently repeated in ‘‘hot’’chemistry conditions, employing the same commercial appa-ratus to verify that the trends in the ‘‘cold’’ chemistry werereflected in the radiochemistry, despite the conditions offluoride generation being different. In this case, the incorpora-tion yield was monitored by both radio-TLC and radio-HPLC.The results, shown in Fig. 5a, demonstrate that the conversionachievable in ‘‘hot’’ chemistry was much greater than in the‘‘cold’’ reactions, due to the large excess of the employedprecursors. However, the trends observed were the same asthose seen with ‘‘cold’’ chemistry, leading to an increasedconversion at higher temperatures. The maximum conversionwas observed at 125 uC, and lower yields obtained at highertemperatures were again considered to be due to decomposi-tion phenomena. Also, the lower conversion at longerresidence times is thought to be due to prolonged exposureof chemicals to high temperatures causing decomposition,

Fig. 4 Optimisation of the ‘‘cold’’ (non-radioactive) radiolabelling reaction. (a)The influence of temperature and residence time of the reagents within themicrochannel on the conversion of mannose triflate 1 to acetylated 19F-FDG 2,and (b) effect of the ratio of 1 to 19F-fluoride (from potassium fluoride) on theconversion.

Fig. 5 Optimisation of the ‘‘hot’’ (radioactive) radiolabelling reaction. (a) Theinfluence of temperature and residence time on the conversion of 1 to 2, and (b)the effect of residence time on the reaction conversion at125 uC.

This journal is � The Royal Society of Chemistry 2013 Lab Chip, 2013, 13, 2328–2336 | 2333

Lab on a Chip Paper

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online

which could not be observed in the ‘‘cold’’ chemistry due tothe low conversions reported. Therefore, a further investiga-tion on residence time influence was conducted, the results ofwhich are reported in Fig. 5b, and show that a residence timeas short as 3 s is sufficient to obtain the same incorporationyield as that achievable in 30 s. Regarding the 10% discrepancybetween the results obtained by radio-HPLC and radio-TLC inFig. 5a–b, this is a phenomenon generally encountered inradiochemistry due to the tendency of HPLC methods tounderestimate the quantity of radioactive 18F-fluoride, due to asmall amount of sticking of this anion onto the C18 column.

The basic hydrolysis (step 5) of 2 to 3 was investigated usingonly ‘‘hot’’ chemistry. A slightly revised microreactor designwas employed here, such that the radiolabelling reaction wasperformed as in the previous design, but with the addition of athird inlet downstream that allowed injection of sodiumhydroxide (0.1 M) into the microreactor (Fig. S3, ESI3). Theradiolabelling conditions (step 3, conversion of 1 to 2) were setto a 12 s residence and a temperature of 125 uC. Of particularnote here was the fact that no solvent exchange process (step4) took place prior to hydrolysis. NaOH was pumped into thethird inlet, and allowed to mix and react with 2 to form 3, withthe 1.5 mL channel positioned over a heating element held.The flow rate of NaOH was varied in order to study the effect ofthe ratio of NaOH : Mannose triflate 1 (the lower the ratio ofNaOH, the longer the residence time), while the temperatureof the channel was also varied. The results are shown in Fig. 6,and demonstrate that higher ratios of NaOH are required toincrease the conversion to product 3. It was found thatconducting the hydrolysis reaction at 20 uC using a 10 : 1NaOH ratio yielded the product in highest selectivity. It shouldalso be noted that if the reaction was conducted at a highertemperature using a 2 : 1 ratio of NaOH, all of the startingmaterial was consumed, but resulted in a number ofbyproducts being formed.

18F-FDG synthesis in the ROC platform

The operations performed in order to synthesise 18F-FDG inthe ROC system are shown in Fig. 7a. In step 1, the pre-

concentration of 18F-fluoride, an aqueous solution (1.5 mL) of18F-fluoride was generated by the cyclotron, and delivered intomodule 1 (pre-concentration chip, Fig. 1) at a flow rate of 500mL min21 via a syringe pump placed inside the hot cell, withthe Auto Valve Switch (2-position, 6-port valve) on the platformplaced in the ‘‘no-production’’ mode. The 18F-fluoride becametrapped on the anion exchange resin present in microfluidicchamber, with the excess 18O-water directed into a waste line.Following this, the Auto Valve Switch was turned to the‘‘production’’ position, initiating the delivery of acetonitrile/K222/K2CO3 solution into module 1, which in turn graduallyeluted the radioactive 18F-fluoride from the resin (effectivelyperforming the solvent exchange procedure, step 2).

The radioactive solution exiting module 1 was directed intoone inlet port of module 2 (radiolabelling chip, Fig. 2), whilean acetonitrile solution of mannose triflate 1 was introducedvia a second inlet. The two reagent streams were allowed to

Fig. 6 Graph showing the effect of the ratio of sodium hydroxide to mannosetriflate 1 on the ‘‘hot’’ hydrolysis of acetylated-18FDG 2 to 18F-FDG 3.

Fig. 7 (a) Schematic illustrating the operational setup of the ROC platform forsynthesising 18F-FDG, including the connection of modules 1 (pre-concentra-tion), 2 (radiolabelling), and 3 (hydrolysis) in relation to each other. (b) Radio-TLC profiles of basic hydrolysis test reactions (conversion of 2 to 3). Theunhydrolysed mixture (containing acetylated-18F-FDG 2) is shown in purple,while the remaining colours show the effect of temperature on the hydrolysisreaction. Peaks due to unreacted 18F-fluoride occurred at 10–20 mm, with 18F-FDG 3 at 40–50 mm, and acetylated-18F-FDG 2 at 70–80 mm.

2334 | Lab Chip, 2013, 13, 2328–2336 This journal is � The Royal Society of Chemistry 2013

Paper Lab on a Chip

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online

react within the serpentine chamber, with the temperaturemaintained via the Minco heater below the microfluidic device,yielding acetylated-18F-FDG 2. Importantly, test radiofluorina-tion reactions performed in this manner demonstrated thatK222/K2CO3/18F-fluoride solution eluted from the pre-concentra-tion module (containing 10% v/v of water) allowed incorpora-tion yields (of 18F-fluoride into 1 to give 2) of up to 81% to beobtained, even without an intermediate azeotropic distillationstep (solvent exchange, step 2). This fact, currently unreported,is leading our group to a more detailed study of water tolerancein microfluidic reactions (paper in preparation). Test reactionswere performed in order to study the effect of different flowratios of the two reagents on the conversion yield. Theoptimised setup showed that maximum incorporation wasachieved at 125 uC when the K222/K2CO3/18F-fluoride solutionwas delivered at 20 mL min21, while the solution of compound 1was delivered at 80 mL min21. However, in the fully integratedprocedure performed on the ROC platform, an overall slowerflow rate was applied in order to reduce backpressure that couldpotentially cause breakages or leakages. Therefore, the flow rateof the 18F-fluoride complex was reduced to 10 mL min21, withthe flow rate of 1 also set to 10 mL min21, and an appliedtemperature of 125 uC. These settings produced a steadyincorporation yield of 62%.

As had been determined by the optimisation of the syntheticsteps on the commercial Labtrix Start system earlier, solventexchange from acetonitrile to water (step 4) was found to not berequired between the radiolabelling and hydrolysis steps. Thisallowed the solvent exchange module to be bypassed in an effortto simplify the procedure, a choice made possible by themodular nature of the platform, enabling the solution of 2 inacetonitrile produced in module 2 to be routed directly into thehydrolysis device (module 4, total volume of 4 mL) via one of thetwo inlet ports (Fig. 7a). Into the second inlet port was fed anaqueous solution of sodium hydroxide (0.1 M), at a flow ratethat was 1 : 5 in favour of the NaOH solution (100 mL min21)compared to the solution of 2 (20 mL min21), corresponding to aNaOH : Mannose triflate ratio of 10 : 1 as had been optimisedearlier. The effect of temperature on the hydrolysis reaction wasinvestigated, with radio-TLC analysis employed to determinethe results (shown in Fig. 7b). It was found that when theprocess was conducted at >30 uC, the presence of 2 vanished,with the appearance instead of a non-moving radioactiveproduct (unidentified). When the hydrolysis microreactor wasnot heated, and instead allowed to operate at a steadytemperature of 30 uC (due to environmental conditions), thedesired product of 18F-FDG 3 was generated with a yield of 60%.

The optimised system was subsequently run with freshbatches of starting radioactivity, in order to evaluate thetiming and robustness of the apparatus. Irradiated 18O-waterwas introduced and the 18F-fluoride was trapped in the pre-concentration module, a process requiring less than 5 min.Following this, the platform was set to ‘‘production’’ mode,whereupon monitoring of the backpressure of the pumpsallowed a steady-state condition to be established within 10min. After this timeframe, the system delivered a continuousproduct solution containing 60 ¡ 3% of 18F-FDG 3, andradioactivity was collected for up to 120 min with aconcentration that decreased following radioactive decay. We

estimated that starting from 5 GBq of radioactivity, it would bepossible to obtain 370 MBq (typical injected activity of 18F-FDGfor human studies) after 19 min of continuous collection ofthe product. This value takes into account the real activityexiting the system and regards the first dose collectible afterthe initial 10 min conditioning period. Successive doses wouldrequire more time, according to the decay characteristics ofthe 18F-fluoride nuclide. The projection of this estimationsuggests that a starting activity of 100 GBq of 18F-fluoride,more than 500 MBq of 18F-FDG would be collected within thefirst minute of production, following the 15 min initialisation(5 min pre-concentration plus 10 min conditioning). Thesystem so far has only been tested with activities up to 5 GBq,and so no definite forecasts can be made on the actualutilisation of higher starting doses.

Off-platform continuous flow purification has been testedwith modules similar to those used for 18F-fluoride pre-concentration. These modules were filled with commerciallyavailable solid-phase extraction resins and aligned in a ‘‘train’’configuration, with fluid from one chip into the next until theproduct solution had been passed consecutively through eachof the resins.16 In these standalone experiments, 18F-FDGmixture from module 4 was diluted on-line with water andpassed through the purification system, with the final radio-tracer being obtained with a radiochemical purity of 86%,though improvements are needed before reaching injectable(>95% r.c. purity) doses. Such improvements could beachieved by the implementation of a final solvent exchangestep15 prior to micro-SPE purification, which would reduce theamount of acetylated-18F-FDG passing through the purificationmodules without being trapped. Hence, while a solventexchange from acetonitrile to water (step 4) is not requiredfor the conversion of acetylated-18F-FDG 2 to 18F-FDG 3 byhydrolysis, it would actually be beneficial for the purificationstep at the end of the synthetic procedure.

Based upon the performance of the ROC platform, and withconsideration of the possible further developments of thesystem, the fluidic technology described here represents asignificant step towards the DOD of 18F-FDG and otherradiopharmaceuticals that require a two-step synthesis. Thehigh level of control, the modular nature of the system thatallows relevant modules to be swapped in and out, and thelarge surface-to-volume ratio of the microreactors should allowvalidation of this system for multiple production routes,within GMP conditions. Considering that many PET imagingcentres currently refrain from production of their own tracers(due to the high cost of cyclotrons and complex productionpractices/regulations), the technology developed here couldprovide commercial producers a unique platform to be usedfor synthesising a wider variety of PET tracers. Moreover, itcould allow research centres that purchase 18F-fluoride to useit for synthesising several types of tracers on the sameapparatus, thus reducing costs.

In conclusion, the ROC platform is capable of producing18F-FDG with similar yields (60%) to traditional batchsynthesisers, while the modular format has several advantagescompared to conventional processes:

i. Production of a single 18F-FDG dose can be collected after19 min, while the entire bolus of radioactivity can be processed

This journal is � The Royal Society of Chemistry 2013 Lab Chip, 2013, 13, 2328–2336 | 2335

Lab on a Chip Paper

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online

continuously for 2 h if further doses are required. Intraditional systems, 18F-FDG is typically obtained after 35–40min, and there is no possibility of collecting a single dose.

ii. The radiolabelling reaction occurs within 60 s ofresidence time inside the microreactor, compared to the 5–10 min needed in vessel radiochemistry.

iii. Reagent consumption is reduced to a minimum both forproducing a single dose, and for processing the wholeradioactive bolus.

iv. Waste production is minimised due to the low volumes ofsolutions involved.

v. Operator interactions are reduced and reaction processesare both simpler and employ lower volumes of radioactivesolutions, leading to safer operation for the user.

vi. The ROC platform has the potential to be adapted to theproduction of other radiotracers that require a two-step syntheticapproach and SPE purification (e.g. 18F-fluorocholines), simply byadjusting the reagents and the order of the modules.

Conclusions

We have developed a modular microfluidic platform for theproduction of radiopharmaceuticals in continuous mode froma single bolus of radioactive solution. The platform consists offour modules that perform the basic operations needed fortypical radiosyntheses. To produce 18F-FDG, we optimised amethod employing the ROC platform based on the use of onlythree modules, thereby simplifying the traditional syntheticroute and obtaining 18F-FDG in rapid time (i.e. within a fewminutes of equilibration time) and with high yields.

Thanks to the modular approach, the ability to simplify the18F-FDG synthetic routes, and the possibility to provide 18F-FDG in a dose-on-demand manner, we believe that the ROCprototype guides the way to routine, high-quality and efficientradiochemical synthesis of standard radiolabelled com-pounds, as well as the generation of new radiopharmaceu-ticals.

Acknowledgements

The authors acknowledge the EU project Radiochemistry onchip ‘‘ROC’’, grant agreement no. 213803 for financial support.

References

1 K. Serdons, A. Verbruggen and G. M. Bormans, Methods,2009, 48, 104–111.

2 G. Pascali, G. Mazzone, G. Saccomanni, C. Manera and P.A. Salvadori, Nucl. Med. Biol., 2010, 37, 547–555.

3 G. Pascali, G. Nannavecchia, S. Pitzianti and P. A. Salvadori,Nucl. Med. Biol., 2011, 38, 637–644.

4 A. M. Elizarov, Lab Chip, 2009, 9, 1326–1333.5 P. Watts, G. Pascali and P. Salvadori, J. Flow Chem., 2012, 2,

37–42.6 J. M. Gillies, C. Prenant, G. N. Chimon, G. J. Smethurst,

W. Perrie, I. Hambletta, B. Dekker and J. Zweit, Appl.Radiat. Isot., 2006, 64, 325–332.

7 P. Y. Keng, S. P. Chen, H. J. Ding, S. Sadeghi, G. J. Shah,A. Dooraghi, M. E. Phelps, N. Satyamurthy, A.F. Chatziioannou, C. J. Kim and R. M. van Dam, Proc.Natl. Acad. Sci. U. S. A., 2011, 109, 690–695.

8 S. Y. Lu, P. Watts, F. T. Chin, J. Hong, J. L. Musachio,E. Briard and V. W. Pike, Lab Chip, 2004, 4, 523–525.

9 C. C. Lee, G. D. Sui, A. Elizarov, C. Y. J. Shu, Y. S. Shin, A.N. Dooley, J. Huang, A. Daridon, P. Wyatt, D. Stout, H.C. Kolb, O. N. Witte, N. Satyamurthy, J. R. Heath, M.E. Phelps, S. R. Quake and H. R. Tseng, Science, 2005, 310,1793–1796.

10 C. J. Steel, A. T. O’Brien, S. K. Luthra and F. Brady, J.Labelled Compd. Radiopharm., 2007, 50, 308–311.

11 A. Lebedev, R. Miraghaie, K. Kotta, C. E. Ball, J. Zhang, M.S. Buchsbaum, H. C. Kolb and A. Elizarov, Lab Chip, 2013,13, 136–145.

12 A. M. Elizarov, R. M. van Dam, Y. S. Shin, H. C. Kolb, H.C. Padgett, D. Stout, J. Shu, J. Huang, A. Daridon and J.R. Heath, J. Nucl. Med., 2010, 51, 282–287.

13 A. Zacheo, V. Arima, G. Pascali, P. Salvadori, A. Zizzari,E. Perrone, L. De Marco, G. Gigli and R. Rinaldi, Microfluid.Nanofluid., 2011, 11, 35–44.

14 F. De Leonardis, G. Pascali, P. A. Salvadori, P. Watts andN. Pamme, J. Chromatogr., A, 2011, 1218, 4714–4719.

15 B. Z. Cvetkovic, O. Lade, L. Marra, V. Arima, R. Rinaldi andP. S. Dittrich, RSC Adv., 2012, 2, 11117–11122.

16 M. D. Tarn, G. Pascali, F. De Leonardis, P. Watts, P.A. Salvadori and N. Pamme, J. Chromatogr., A, 2013, 1280,117–121.

17 T. McCreedy, TrAC, Trends Anal. Chem., 2000, 19, 396–401.

2336 | Lab Chip, 2013, 13, 2328–2336 This journal is � The Royal Society of Chemistry 2013

Paper Lab on a Chip

Publ

ishe

d on

25

Mar

ch 2

013.

Dow

nloa

ded

by U

nive

rsita

del

Sal

ento

on

23/0

5/20

14 0

7:52

:27.

View Article Online