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Cite this: DOI: 10.1039/c9lc00676a

Received 13th July 2019,Accepted 15th August 2019

DOI: 10.1039/c9lc00676a

rsc.li/loc

Liquid marbles as biochemical reactors for thepolymerase chain reaction†

Kamalalayam Rajan Sreejith,a Lena Gorgannezhad,ab Jing Jin, a Chin Hong Ooi,a

Helen Stratton,b Dzung Viet Daoa and Nam-Trung Nguyen *a

The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Tech-

nological and engineering advancements in the field of microfluidics have fuelled the progress of polymer-

ase chain reaction (PCR) technology in the last three decades. Advances in microfluidics-based PCR tech-

nology have significantly reduced the sample volume and thermal cycling time. Further advances led to

novel and accurate techniques such as the digital PCR. However, contamination of PCR samples, lack of

reusability of the microfluidic PCR platforms, complexity in instrumentation and operation remain as some

of the significant drawbacks of conventional microfluidic PCR platforms. Liquid marbles, the recently

emerging microfluidic platform, could potentially resolve these drawbacks. This paper reports the first liquid

marble based polymerase chain reaction. We demonstrated an experimental setup for the liquid-marble

based PCR with a humidity-controlled chamber and an embedded thermal cycler. A concentrated salt so-

lution was used to control the humidity of the PCR chamber which in turn reduces the evaporation rate of

the liquid marble. The successful PCR of microbial source tracking markers for faecal contamination was

achieved with the system, indicating potential application in water quality monitoring.

1. Introduction

The polymerase chain reaction (PCR) has been emerging as apopular and powerful amplification technique ofdeoxyribonucleic acids (DNAs) since its invention in 1985.1 Ina PCR, a mixture containing template DNAs, primers, DNApolymerase, deoxyribonucleotide triphosphates (dNTPs) andbuffer solution is subjected to a series of thermal cycles toyield millions of copies of template DNA.2–5 End-type PCR,quantitative PCR (qPCR) and digital PCR (dPCR) are the threewell established, optimised and widely used PCR techniques.The outcome of an end-point PCR is evaluated at the end ofthe reaction using a separate gel electrophoresis device, whilethe outcome of a qPCR and dPCR is evaluated using the fluo-rescence signal emitted from the reaction mixture upon suc-cessful DNA amplification. These PCR techniques have theirown advantages, disadvantages and specific applications.5

However, PCR generally finds applications in biotechnology,genetic engineering, cell biology, forensic science, water re-search, drug discovery research, food micro-biology, etc.6–12

Advances in microfluidics technology in the last three de-cades have revolutionised the implementation of PCRtechnology.5,13–16 Many research papers were published anddemonstrated PCR carried out in various microfluidic plat-forms including droplet,17,18 micro well,19–21 micro chan-nels,22,23 etc. However, these methods are limited by the vul-nerability to contamination, lack of reusability of theplatform and the requirement for sophisticatedinstrumentation.

Despite the advanced microfluidics-based PCR technolo-gies, the use of liquid marbles as biochemical reactors forPCR has not been reported in the literature to the best of ourknowledge. Liquid marbles are liquid droplets covered by athin layer of hydrophobic particles.24 Conventional liquidmarbles are generated by placing and rolling a liquid dropletover a hydrophobic powder bed. The hydrophobic powder coat-ing avoids physical interaction between the liquid and the am-bient atmosphere, preventing the possibility of contamination.The production25 and manipulation26–37 of liquid marblesare recently relatively well developed. Liquid marbles remainmechanically robust even under relatively high mechanicalstress.38–41 One of the major concerns needing attention isthe non-recyclable plastic waste produced by bioscience labo-ratories across the world. Approximately 5.5 million tons ofplastic waste was estimated to be generated from laboratoriesaround the world in 2014.42 Liquid marbles utilise only a thinhydrophobic powder to encapsulate the liquid droplet, thus

Lab ChipThis journal is © The Royal Society of Chemistry 2019

aQueensland Micro- and Nanotechnology Centre, Griffith University, 170 Kessels

Road, 4111 Queensland, Australia. E-mail: nam-trung.nguyen@griffith.edu.aub School of Environment and Science, Nathan Campus, Griffith University, 170

Kessels Road, 4111 Queensland, Australia

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9lc00676a

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eliminating the usage of disposable plastic consumables forcarrying out biochemical reactions. The potential of liquidmarbles for “green bio-laboratory” practices are still to be ex-plored. These advantages make liquid marbles an ideal can-didate to be used as bioreactors to carry out biological andbiochemical reactions.

Arbatan et al. demonstrated rapid blood typing in a liq-uid marble.43 Sarvi et al. used liquid marbles for cardiog-enesis of embryonic stem cells.44 Ledda et al. successfullydemonstrated in vitro maturation of sheep oocytes inside aliquid marble.45 Vadivelu et al. successfully used liquidmarbles as bioreactors for cryopreservation of cells.46 De-spite the vast usage of liquid marbles as bioreactors forvarious biological and biochemical reactions, the potentialof liquid marbles as bioreactors for the polymerase chainreaction has never been explored. To the best of our knowl-edge, no research paper has reported the usage of liquidmarbles for PCR.

The fundamental difficulty in using liquid marbles aspractical reactors for PCR is the evaporation of the reagentmixture during thermal cycling.47 The evaporation process ofdifferent liquid marble types under various physical condi-tions was extensively studied and reported in theliterature.48–51 Our previous studies had provided insightsinto the different parameters affecting the evaporation rate ofliquid marbles.47 In the present work, a customised experi-mental setup was developed to minimize the evaporation ofliquid marbles during the PCR experiments. The present pa-per reports the first successful PCR using a liquid marble asa biochemical reactor.

2. Materials and methods

Fig. 1a shows the schematic of the experimental setup.Fig. 1b demonstrates the exploded view of the custom-madeliquid marble PCR chamber with an embedded thermal cy-cler. Fig. 1c is the photograph of the experimental setup. De-tailed description of the fabrication of the liquid marble PCRchamber and thermal cycler is provided in the followingsubsections.

2.1 Preparation of the polymerase chain reaction mixture

Aiming at application in water quality monitoring, DNA wasextracted from fecal samples of a healthy individual using aQIAamp DNA Stool Mini Kit (Qiagen) according to the manu-facturer's protocol and was stored at −20 °C. A primary ampli-fication of the extracted DNA was carried out in a conven-tional PCR machine using the GoTaq Green master mix(Promega). Reverse primer sequence: 5′CGTTACCCCGCCTACTATCTAATG-3′ and forward primer sequence: 5′-TGAGTTCACATGTCCGCATGA-3′ were used for the detection of thehuman specific faecal DNA marker (BacHuman). The reactionmixture (20 μl) contained 10 μl of GoTaq Green master mix,2.5 μl (10 μM) of each forward and reverse primer, and 2.5 μlof the template DNA and 2.5 μl of DNA free water. The PCRwas performed under the following conditions: initialannealing at 50 °C for 2 min, 95 °C for 10 min, followed by39 cycles at 95 °C for 15 second and 60 °C for 1 min. Then,the resulting template DNA was used for DNA and primer op-timization (provided in the ESI† S1). Serial dilutions of thetemplate DNA (25–400 ng μl−1) were carried out and used inthe subsequent experiments.

Fig. 1 Liquid marble based PCR: a) experimental setup; b) exploded view of the PCR chamber and thermal cycler assembly; c) photograph of theexperimental setup.

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Five 50 μl PCR mixture samples were prepared in dupli-cate by mixing the template DNA, forward and reverse primersequences and SsoFast EvaGreen Supermix (Bio-Rad). Thesample set contained 400 ng μl−1, 200 ng μl−1, 100 ng μl−1, 50ng μl−1, and 25 ng μl−1 template DNA concentrations. Onesample set was subsequently used for liquid marble PCR ex-periments, and the other sample set was used to carry outthe conventional qRT-PCR. A negative control for BacHumanwas prepared using S. aureus DNA.

2.2 Preparation of PCR mixture liquid marbles

Liquid marbles made of the PCR mixture were formed by dis-pensing the PCR mixture liquid droplets on a bed of poly-tetrafluoroethylene (PTFE) powder (Sigma-Aldrich, 1 μmnominal diameter, ρ = 2.2 g cm−3) and subsequent rolling. Amicropipette (model CH 02607 by Thermo Electron Corpora-tion) was used for droplet dispensing as it provides good con-trollability on the volume and the diameter of the droplet.The PTFE powder used for the experiment was sterilised for20 minutes in an ultraviolet irradiation chamber. Liquid mar-bles with 50 μl volume were prepared and used in theexperiments.

2.3 Design and fabrication of the thermal cycler

A customised thermal cycler was developed to carry out thepolymerase chain reaction in a liquid marble coated withPTFE powder. A 20 × 20 × 15 mm3 aluminium block embed-ded with a 30 W cylindrical cartridge heater (Core Electron-ics) was used as the heating platform of liquid marbles. AnLM 35 semiconductor temperature sensor was mounted onthe aluminium heater block for measuring the temperature.The heating platform was mounted on a 40 × 40 × 3.5 mm3

thermoelectric Peltier cooler module (TEC-12706 AUSElectronics) using a heat conductive adhesive (Stars-922 heatsink plaster). The Peltier module was subsequently mountedon an aluminium heat sink (85.4 × 68.3 × 41.5 mm3) of acomputer CPU cooler fan (12 V, 3300 rpm, 70 × 70 × 25 mm3)using a heat conductive adhesive. The cartridge heater andthe Peltier cooler modules were controlled by a PID controlalgorithm implemented in an Arduino Mega microcontrollerboard. The proportional, integral and derivative gains wereoptimized to obtain faster heating and cooling with mini-mum overshooting and steady-state error. The smaller size ofthe heating platform also helped to reduce thermal inertiaand achieve a faster thermal response. The temperatureramping rate of our customised thermal cycler was 0.68 K s−1

during heating and 1 K s−1 during cooling.A dummy experiment was carried out to check the thermal

response of the liquid marble placed on the thermal cycler.The Arduino microcontroller was programmed to heat the al-uminium block to 75 °C. A 50 μl water liquid marble wasplaced on the aluminium heating block. A calibrated negativetemperature coefficient (NTC) thermistor (Build Circuit, Aus-tralia) was vertically inserted into the liquid marble using aprecision positioning stage. The temperature of the liquid

marble during ramping and steady state was recorded in realtime. Fig. 2 compares the temperatures of the aluminiumheater block and of the liquid marble. The inset shows a liq-uid marble with an NTC thermistor. The liquid marble tem-perature closely followed the heater block temperature withan average difference of only 1.24 ± 0.62 K. This experimentconfirms reasonably good heat transfer from the heater blockto the liquid marble.

2.4 Design and fabrication of the polymerase chain reactionchamber

The primary difficulty in using liquid marbles as a micro-reactor to carry out the PCR is the evaporation of the PCRmixture through the porous PTFE coating. We alreadyreported in a previous study that high ambient humidity re-duces the rate of evaporation of a liquid marble at elevatedtemperatures.47 Hence, a humidity controlled chamber wasdeveloped to carry out the liquid marble based polymerasechain reaction. A 100 × 100 × 60 mm3 rectangular airtightplastic container was used to make the humidity-controlledPCR chamber. The heating platform of the thermal cyclersubsystem was inserted into the container through a speciallymade rectangular opening at the bottom of the container.Proper sealing at the bottom of the container ensured air-tightness. A rectangular opening was made on the top coverof the plastic container at a position vertically above theheating platform. A clear glass plate was used to close thisopening and proper sealing was ensured for air isolation.The clear glass helps to obtain better visibility of the liquidmarble lying on the heating platform.

An additional cartridge heater module was affixed on theouter side of the clear glass using a heat conductive adhesive.This cartridge heater could be turned on to avoid vapour con-densation on the glass plate during the thermal cycling pro-cess of the liquid marble. The condensation would reducethe visibility of the liquid marble. An 8 × 5 mm2 rectangular

Fig. 2 Comparison of aluminium block temperature and liquid marbletemperature. The inset picture shows the photograph of the liquidmarble inserted with a temperature sensor.

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openable window was made at the side of the plastic reactionchamber. This window is used to place the liquid marble onthe heating platform before starting the thermal cyclingprocess.

A well-known method52 with a saturated salt solution wasused to control the relative humidity inside the reactionchamber. A saturated solution of potassium sulfate was pre-pared and subsequently kept inside the reaction chamber in6 separate rectangular plastic dishes (35 × 35 × 8 mm3). Thereaction chamber was kept closed for 16 hours for humiditystabilisation. The humidity inside the chamber was continu-ously monitored using a humidity sensor (Senonics Minnow)positioned inside the reaction chamber. The average relativehumidity of the reaction chamber over the duration of the ex-periment was 96.3 ± 0.7%.

2.5 Design and fabrication of the fluorescence detectionsystem

The fluorescence excitation wavelength of the proposed PCRmixture is between 450 and 490 nm (blue light) and it isexpected to provide an emission spectrum between 520 and560 nm (green) upon successful DNA amplification. Theintensity of the emitted green light is the indicator of amplifi-cation efficiency. A blue light emitting diode (LED) (450–490nm) served as the source of fluorescence excitation. An LEDlighting system used in this experiment was custom-builtwith a concentric circular arrangement of 30 blue LEDs (1500mCd, Jaycar, Australia). The green fluorescent light emittedfrom the liquid marble PCR microreactor was captured usinga vertically mounted CMOS camera (Edmund Optic EO-5012C) attached with a 0.5× telecentric lens (Edmund Optics-63074). The light emitted from the liquid marble was filteredusing a green optical filter (520–560 nm) before being cap-tured using the camera to improve the signal to noise ratio.

2.6 Experimental procedure

The synthesized PCR mixture liquid marble (sections 2.1 and2.2) was transferred to the heating platform with a steel spat-ula through an openable window made on the sidewalls ofthe PCR chamber. The window was closed after successfulplacement of the liquid marble. The liquid marble wasplaced vertically below the camera so that a clear image canbe captured. The heater mounted on the top of the glass win-dow was turned on and the glass window was heated to pre-vent possible vapor condensation during the thermal cyclingprocess of the liquid marble. Heating of the glass window en-sured better visualisation of the liquid marble during thethermal cycling process.

The thermal cycling subsystem was subsequently turnedon. The thermal cycler was programmed in such a way thatthe liquid marbles underwent a onetime initiation process at95 °C for 15 seconds. The liquid marble was subsequentlysubjected to denaturation at 95 °C for 15 seconds and a com-bined annealing and extension process at 60 °C for 40 sec-onds. The thermal cycle was repeated for 15 minutes. Fig. 3a

shows the temperature characteristics of the thermal cycler.Fig. 3b shows the relative humidity of the PCR chamber dur-ing the period of experiment. The thermal cycler exhibited amaximum overshoot of +8 K from the upper temperaturesetpoint (95 °C) during the first thermal cycle and stabilisedto steady state in subsequent cycles. The temperature of theblock read 95 ± 0.7 °C and 60 ± 1.5 °C during the steady-statethermal cycles. The recorded video of the liquid marble dur-ing the thermal cycling process was captured throughout. Avolume of 50 μl PCR mixture with DNA concentrations vary-ing from 25 ng μl−1 to 400 ng μl−1 was used for the experi-ments. Experiments were repeated three times.

Thermal cycling of the 50 μl PCR mixture (five sampleswith DNA concentrations varying from 25 ng μl−1 to 400 ngμl−1) was carried out in a conventional qRT-PCR machine(Bio-Rad CFX connect) for 15 minutes with similar thermalcycling conditions explained earlier.

3. Results and discussion

Thermal cycling of the liquid marbles containing the PCR re-action mixture was carried out as explained above in section2.6. Fig. 4a shows the representative fluorescence intensityemitted from the liquid marbles with various DNA

Fig. 3 Parameters of the PCR using a liquid marble: a) thermal cyclesof the PCR machine. b) Variation of humidity inside the PCR chamberduring the experiment.

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concentrations. The numerical equivalent values of fluores-cence intensities of the liquid marbles at various time inter-vals were evaluated using ImageJ53 open source software. Thefluorescence intensity values of liquid marbles were offsetcorrected and normalised subsequently. Offset correctionand normalisation of fluorescence intensities were done asfollows:

Ist_Normalised = (Ist − Is0)/IMax (1)

where Ist is the fluorescence intensity of a sample mea-sured at a given time, Is0 is the fluorescence intensity ofthat sample at the beginning of thermal cycling and IMax isthe maximum fluorescence intensity recorded among allthe samples in the experiment. Fig. 4b shows the changeof the normalised fluorescence intensities over time of liq-uid marbles with different DNA concentrations. The resultsindicate that the fluorescence intensity of liquid marblescontaining the PCR mixture with different concentrationsincreased over time up to a certain time point and subse-quently decreased. The increase of fluorescence intensity ofthe PCR mixture liquid marbles indicates the positive poly-merase chain reaction inside the liquid marbles. The liquidmarble containing the negative sample of the PCR mixturewas not giving any significant fluorescence on thermal cy-cling. This proves the specificity of the PCR inside the liq-uid marble. Maximum fluorescence was observed during aperiod of 11–12 minutes (corresponding to 9 thermal cy-cles) for all the liquid marbles. We observed that the fluo-rescence intensity of the liquid marbles reduced afterreaching a maximum value.

Fig. 4c shows the comparison of normalised fluorescenceintensities of reactions carried out in a conventional qRT-PCR machine (Biorad CFX Connect) and that of a real-timeliquid-marble PCR. We observed that the trends in maximumfluorescence intensities emitted by the samples in both casesare similar. The optimum DNA concentration for maximumfluorescence intensity was observed to be 100 ng μl−1 in bothexperiments. The fluorescence intensity variation with respectto the DNA concentration follows a similar pattern in bothexperiments. These results confirm the successful polymerasechain reaction and amplification of template DNA inside aliquid marble.

We hypothesize that the reason for the deterioration offluorescence is photobleaching of fluorescent dye due to con-stant illumination. The fluid medium of the PCR mixture wasobserved to be completely evaporated after 12 minutes ofthermal cycling, resulting in no further DNA amplification.Continuous illumination of the sample with blue light maycause photobleaching and can result in deterioration of fluo-rescence intensity over time.

A separate experiment was carried out to test the possibil-ity of photobleaching. A volume of 50 μl of PCR sample mix-ture with 100 ng μl−1 template DNA underwent 20 thermal cy-cles in a conventional PCR machine (Biorad CFX Connect). Aliquid marble is made out of this DNA amplified sample andkept under the fluorescence detection system described insection 2.5. A continuous video of the fluorescence emittedfrom the liquid marble is captured for 15 minutes. The exper-iment was repeated three times. The fluorescence intensity ofthe liquid marble at regular time intervals was evaluatedusing ImageJ software. An average deterioration of 22.5% in

Fig. 4 PCR results: a) micrographs showing the fluorescence from PCR mixture liquid marbles with different DNA concentrations at variousintervals of thermal cycling. b) Variation of fluorescence intensity of PCR mixture liquid marbles with respect to time during thermal cycling. c)Comparison of normalised peak fluorescence intensities of the liquid marble PCR and conventional qRT-PCR at the time of maximum fluorescenceemission from the corresponding liquid marble PCR sample.

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fluorescence intensity was observed over a period of 15 mi-nutes of continuous illumination. Fig. 5 shows the decreasein magnitude of fluorescence intensity with respect to time.This decrease in intensity verifies that the fluorescence dete-rioration of the liquid marble PCR after reaching a maximumfluorescence intensity is due to photobleaching of the dye un-der continuous illumination.

4. Conclusions

We designed and developed a customised thermal cycler toconduct the liquid marble-based polymerase chain reaction.A humidity-controlled chamber was developed to reduce therate of evaporation of the PCR mixture from the liquid mar-ble during the thermal cycling process. Successful amplifica-tion of DNA extracted from fecal samples of a healthy personwas carried out by the real-time polymerase chain reactionusing the PTFE liquid marble as a bioreactor.

The fluorescence emitted from the liquid marble was cap-tured in real time using a camera. The fluorescence intensityemitted from liquid marbles containing various DNA concen-trations was plotted as a function of time. The optimum DNAconcentration for efficient amplification was 100 ng μl−1. Thisoptimum DNA concentration for efficient amplificationobtained from the liquid marble-based PCR was confirmedwith a conventional qRT-PCR experiment conducted in acommercial machine (Biorad CFX Connect). The trend in var-iation of fluorescence intensities with template DNA concen-tration was also observed to be similar in both the liquidmarble PCR and conventional qRT-PCR.

Evaporation of the PCR mixture through the porous wallsof the liquid marbles limits the duration of thermal cyclingand hence the amplification efficiency of the proposedmethod. Deterioration of fluorescence was observed in theliquid marble-based PCR after a certain number of thermalcycles. Photobleaching of the fluorescent dye was found to bethe reason for the deterioration of fluorescence.

The optimum detection sensitivity achieved with our pres-ent work was 25 ng μl−1. Highly optimised conventional qPCRmachines demonstrate detection sensitivity on the order ofpg μl−1. The relatively lower detection sensitivity of the pres-ent work can be attributed to three major reasons: (i) trans-parency of the hydrophobic powder used in the experiment;(ii) premature evaporation of the liquid marbles and (iii) sen-sitivity and resolution of the optical detection system. Ourwork utilised PTFE powder as the hydrophobic coating. Theoptical transparency of the PTFE coating is worse comparedto other transparent hydrophobic powders reported in sometudies.54,55 Utilising a truly transparent coating may greatlyenhance the optical detection characteristics of liquidmarble-based PCR technology. Transparent liquid marbleswill improve the detection limit of PCR amplification. Thepresented liquid marble-based polymerase chain reactioncould only undergo 9 thermal cycles before gettingcompletely evaporated. An optimised liquid marble technol-ogy with a lower rate of evaporation of the sample would sig-nificantly increase the amplification efficiency and detectionlimit. A hydrophobic powder material with controllablecrosslinking could potentially solve the problem of evapora-tion during the thermal cycling process. Furthermore, our ex-periments utilised a commercial off-the-shelf CMOS camerafor the detection of fluorescence signals. This camera is infe-rior to highly sensitive and optimised fluorescence detectionsystems (photodiodes, photomultiplier tubes, charge coupleddevices, etc.) of conventional qPCR machines. In a nutshell,the detection limit of liquid marble-based PCR technologycan be optimised with optically more transparent coatingpowder, evaporation prevention and highly sensitive fluores-cence detection.

In conclusion, this paper reported the successful PCR oftemplate DNA using a PTFE liquid marble as a biochemicalreactor. The detection sensitivity of the present work is rela-tively lower compared to conventional commercial qPCRmethods due to limited transparency of the hydrophobiccoating, evaporation of the liquid marble and the low sensi-tivity of the camera used in our experiments. All these issuescan be addressed in future studies. The optimized liquid-marble-based PCR is expected to open a new era for the real-time PCR as well as the digital PCR. Future studies will alsobe extended to explore the potential of liquid marbles as abioreactor for the loop mediated isothermal amplification(LAMP) technique, which requires a lower working tempera-ture than that of the PCR. The isothermal low-temperatureLAMP process is expected to reduce the evaporation rate ofthe liquid marble, thereby could provide a comparable per-formance with existing conventional LAMP techniques.

Ethical approval

All experiments were performed in compliance with relevantlaws or guidelines. All experiments followed the guidelines ofGriffith University. Ethical approval for the study on the effec-tiveness of molecular assays in detecting human faecal

Fig. 5 Deterioration of fluorescence intensity of the liquid marble PCRsample under continuous illumination.

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pollution and the effect of freezer storage on human faecalsamples was done by the ethics committee through GriffithUniversity Office of Research, ref no. BPS/01/13/HREC. Nohuman subject has been used in the experiment and in-formed consent was obtained.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge the Australian Research Council forfunding support through the grant DP170100277. This workwas performed in part at the Queensland node of the Austra-lian National Fabrication Facility, a company established un-der the National Collaborative Research Infrastructure Strat-egy to provide nano- and micro-fabrication facilities forAustralia's researchers.

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