SUPPORTING INFORMATION

3D printed fluidic platform with in situ covalently immobilized polymer

monolithic column for automatic solid-phase extraction

Enrique Javier Carrasco-Correa1*, David J. Cocovi-Solberg2, José Manuel Herrero-

Martínez1, Ernesto Francisco Simó-Alfonso1, Manuel Miró*2

1 University of Valencia, Spain, Department of Analytical Chemistry, University of

Valencia, C/Doctor Moliner 50, 46100 Burjassot Valencia 2 FI-TRACE group, Department of Chemistry, University of Balearic Islands, Carretera

de Valldemossa, km 7.5, E-07122 Palma de Mallorca, Spain

19 pages

6 figures

2 tables

*Corresponding authors:

Dr. Enrique Javier Carrasco-Correa

e-mail: enrique.carrasco@uv.es

Tel.: +34963544062

Fax: +34963544436

Prof. Manuel Miró

e-mail: manuel.miro@uib.es

Tel: +34 971172746

Fax: +34 971173426

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1. Experimental Section

1.1. Reagents and standard solutions

All reagents were of at least analytical reagent grade. Highly pure deionized water was obtained

from a Milli-Q system (resistivity > 18.2 MΩ·cm, Merck-Millipore, Molsheim, France). All

labware used along this work was made of glass or PTFE. Stock solutions of target analytes

were prepared from solid analytical reagent grades of 4-hydroxybenzoic acid (HBA),

methylparaben (MP), phenylparaben (PhP), bisphenol A (BPA) and triclosan (TCS) (Merck

KGaA, Darmstadt, Germany) in acetonitrile (ACN), which were step-wise diluted in water for

further assays. 2-propanol (ACS Basic, Scharlau) was used for cleaning of the 3D

stereolithographic prints. Glycidyl methacrylate (GMA), ethylene dimethacrylate (EDMA), 3-

(trimethoxysilyl)propyl methacrylate (γ-MPS) and benzophenone (BZP) were purchased from

Merck KGaA. Azobisisobutyronitrile (AIBN) was obtained from Fluka (Buchs, Switzerland).

Cyclohexanol, 1-dodecanol, dimethyl sulfoxide (DMSO), HPLC-grade ACN, methanol

(MeOH) and acetic acid (HAcO) were from Scharlau. Hexamethylenediamine (HMA), sodium

borohydride, calcium chloride, sodium tetraborate, 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDC) and N-hydrosuccinimide (NHS) were obtained from

Fisher Scientific (Whaltman, USA).

Saliva samples were collected after mouthwash from two healthy patients aged > 18 years, who

signed a written informed consent. The individuals performed a mouthwash with tap water

about fifteen minutes after lunch for 1 min, followed by using a commercial mouthwash (Lacer)

for 3 min. A final mouthwash with tap water was done for about 1 min. After 10 min, the

tongue was positioned touching the palate for about 30-60 s to obtain ca. 1 mL of saliva. The

sample collected was weighed, diluted 1:2 with 0.1% (v/v) acetic acid, and filtered before

analysis by the online extraction system. This research project was approved by the Research

Ethics Committee of the Balearic Islands (ID no. IB 3776/18 PI).

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1.2. Instrumentation

A high-performance liquid chromatographic (HPLC) module system (Jasco, Tokyo, Japan)

controlled by the ChromNAV 2.0 software was used throughout. The chromatographic setup

integrates (i) a PU-4180 RHPLC pump that endures pressures up to 700 bar, (ii) an AS-4050

HPLC autosampler equipped with a high pressure 6-port rotary injection valve furnished with

1/32” i.d. PEEK loop of 50 µL, (iii) a GECKO 2000 column heater, (iv) an MD-4017 photo

diode array detector, and (v) an Onyx monolithic HD-C18 analytical column (100 × 4.6 mm,

Phenomenex, Torrance, CA, USA) preceded by a security guard column Onyx monolithic C18

guard cartridge (5 × 3 mm, Phenomenex). Operational experimental conditions are shown in

Table S2.

The liquid driver of the micro-flow injection system consisted of an Xcalibur syringe pump

(Tecan, Männeford, Switzerland) equipped with a 1.0-mL gastight glass syringe (Hamilton,

Bonaduz, Swizerland), and a 9-position multiport valve on its head. The flow manifold was

furnished with FEP tubing (IDEX, Oak Harbor, USA) (1/16” o.d., 1/32” i.d.). An AIM3200

autosampler (Aimlab, Virginia, Australia) furnished with two 60-position, 12 mL-sample tube

racks were connected to the head valve system for automatic sample switching and setup

conditioning.

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1.3. Unsupervised synchronization of the automatic SPE procedure with the HPLC

separation

The connections of the injection valve of the HPLC were displaced 60° counterclockwise, so

that the valve remained in the load position throughout the HPLC run except during the

‘injection delay’ time (10 s), scripted in the manufacturer’s software (Jasco ChromNAV). The

samples were introduced in the sample info list of the HPLC software, along with an initial

blank injection, and the sequence was launched. When the first sample (blank) is injected, the

injection signal of the autosampler was caught by a digital input of the Cavro XCalibur pump

that triggered the exit of a ‘while’ loop in the CocoSoft 4.4 smart method. Then, the automatic

processing of the next sample occurred concurrently with the HPLC separation of the first

sample (blank) and ended by parking the eluate of the next sample in the injection coil (valve in

load position). The low-pressure fluidic setup method waited again for the next injection signal.

When the HPLC separation is finished, the HPLC valve turned to the injection position,

allowing the mobile phase to displace completely the content of the injection loop into the

column head (10 s). Finally, the HPLC valve was switched again to the load position and

triggered the next automatic sample preparation protocol by the flow-based system. A flow

diagram of the synchronization of the fluidic sample processing system and the HPLC setup is

provided in Fig S2.

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1.4. Alternative reactions and mechanisms for covalent attachment of the organic

monolith to 3D printed devices

First, the 3D-SLA printed fluidic objects were filled with 1 mol L-1 HCl for 1 h at 60 ºC to

convert the acrylate ester moieties from the photopolymerized resin into carboxylic acids. Then,

a 20% γ-MPS in IPA solution was introduced into the 3D printed channel device to react for 1 h

at 25 ºC to allow the carboxylic groups to react with the organosilane and generate pendant

vinyl groups[1]. An alternative procedure consisted of changing the first step by alkaline

hydrolysis with 2 mol L-1 NaOH overnight at 45 ºC followed by cleaning with 0.1 mol L-1 HCl.

In both cases, after UV-polymerization, no covalent attachment of the organic monolith to the

walls of the 3D printed objects was observed, since the sorptive phase came out when it was

flushed with IPA at a relatively low flow rates.

In another approach, the pendant ester groups on the 3D printed materials reacted with a

solution of 0.6 mol L-1 NaBH4 and 0.3 mol L-1 CaCl2 in tetrahydrofuran-IPA (1:2 v/v) for 1 h at

25 ºC[2,3]. The idea behind was to reduce the ester group to alcohol, which was followed by

reacting with 20% γ-MPS in IPA solution for 1 h at 25 ºC. However, as in the case described

above, the monolithic phase was not covalently attached to the walls of the 3D prints.

An interesting alternative is the so-called photografting technique[4] on account of the UV

transparency of the SLA Clean Resin. For this purpose, the 3D printed channel was filled with

5% BZP solution in IPA for 60 min under 16W of low pressure Hg lamp UV radiation[5–7].

After cleaning with IPA, the channel was filled with 15% EDMA in IPA for another 60 min

under UV radiation. Unfortunately, after polymerization, the monolith was not covalently

attached.

Finally, two additional methods involving the generation of amide moieties[6,8–10] were tested.

The 3D printed fluidic object reacted with 2 mol L-1 NaOH for 24 h at 45 ºC. Then, the channel

was washed with 0.1 mol L-1 HCl in water. In the first method the hydrolyzed ester moieties

reacted with 200 mM EDC and 520 mM HMD in water for 2 h at 37 ºC, while reaction with 520

mM HMD in water for 2 h at 100 ºC was used in the latter. In both cases, the resulting amino

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pendant moieties reacted with GMA for 2 h at 60 ºC. In any case, no attachment of the

monolithic structure was observed.

In the synthetic routes describe above, the materials were previously cleaned with the

appropriate solvent, IPA, and then dried by a nitrogen steam after every individual step. All the

reactions were also monitored with ATR-FTIR by 3D-printing cube-shaped structures in order

to perform the various reactions over the surface for further IR spectroscopic analysis.

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1.5. Preparation and characterization of the monolithic columns

The as-modified with vinyl ester groups 3D printed fluidic scaffolds were filled with a

polymerization mixture as described in a previous study11 for in-situ formation of porous

polymer monoliths. In brief, the polymerization mixture consisted of a functional monomer,

GMA, a crosslinker, EDMA, a mixture of porogens, 1-propanol and 1,4-butanediol, and an UV-

initiator, DMPA. The column was carefully closed with blind nuts and UV-photopolymerized

for 16 h (see Experimental section for more details).

The parent monolith was used for comparison purposes against the one decorated with AuNPs.

To fabricate the latter, the parent monolith was chemically modified using 4.5 mol/L ammonia

and the resulting monolith with amino terminated moieties allowed the attachment of the

AuNPs as described elsewhere[11,12]. Fig. S4 shows the characteristic red-garnet color of the

AuNP-modified porous monolith integrated in the 3D printed device. The SEM micrograph

showed the abundance of AuNPs (randomly distributed white spots) covering the monolith

surface. The Au content in the hybrid monolithic material was estimated by UV–vis

spectroscopy, as described in the following: The methacrylate material was polymerized in a

non-modified 3D-printed device and pulled out from the 2.0 mm ID channel by positive

pressure at a flow rate of 2 mL min-1. After drying at 60 ºC for 24 h, the AuNP-modified

monolith was first calcinated at 550 °C for 1 h. The remaining product was mixed with freshly

prepared aqua regia, which was subsequently heated until removal of the nitrogen dioxide gases.

After cooling to room temperature the solution was adequately diluted with 1 mol L -1 HCl and

was subjected to UV–vis analysis. The colorimetric determination of Au was based on the

formation of the bromoaurate ion obtained by mixing chloroauric acid from the sample with

potassium bromide[11]. Absorbance measurements were performed at 380 nm. A calibration

curve (1–20 μg mL−1 Au) was constructed in excess of NaBr (4% w/v). The experimental results

revealed a content of 2.4 wt% Au attached to the polymerized monolithic column.

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Fig. S1. Snapshot of the 3D CAD model to serve as on-line SPE scaffold.

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Fig S2. Flow diagram of the unsupervised synchronization between the HPLC run and the

automatic fluidic sample preparation system using the Cocosoft freeware. The automatic sample

preparation protocol is shown for a sample volume of 200 µL, although it can be easily changed

to increasing loading volumes (e.g., the method will last 23 min for a sample volume of 2.5

mL).

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Fig. S3. ATR-FTIR spectra of the pristine 3D printed fluidic platform and after the various

functionalization reactions: without modification (A), modification with 2 mol L-1 NaOH (B),

reaction with EDC and NHS followed by covalent linkage with HMD (C), and generation of

pendant vinyl groups by using GMA (D).

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Fig. S4. Photograph of the AuNP-modified monolithic phase covalently attached to the 3D-

printed fluidic device and its corresponding SEM micrograph (at 120,000 × magnification).

White spots are AuNPs.

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0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10

Rete

ntion

(%)

Loading volume (mL)

4-HBP - GMA column TCS - GMA column

4-HBP - amino functionalized column TCS - amino functionalized column

4-HBP - AuNPs column TCS - AuNPs column

Fig. S5. Breakthrough volume for 4-HBP (dashed line and square) and TCS (normal line and

circle) expressed as the retention efficiency of the target model analytes loaded at different

concentration levels (equal mass: 1 µg) using covalently immobilized monoliths in 3D-printed

devices using automatic SPE with off-line detection. Parent monolith (black), amino-

functionalized column (blue) and AuNP-modified monolith (red).

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0

10

20

30

40

50

60

70

80

90

100

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Rete

ntion

(%)

mg g-1 sorbent

4-HBP - GMA column TCS - GMA column

4-HBP - amino functionalized column TCS - amino functionalized column

4-HBP - AuNPs column TCS - AuNPs column

Fig. S6. Loading capacity for 4-HBP (dashed line and square) and TCS (normal line and

circle) expressed as the retention efficiency of the target analytes loaded at different

concentration levels (equal sample volume: 200 µL) using covalently immobilized

monoliths in 3D-printed devices using automatic SPE with off-line detection. Parent

monolith (black), amino-functionalized column (blue) and AuNP-modified monolith

(red).

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Table S1. Operational procedure of automatic flow-through sorptive microextraction procedure for a sample volume of 0.2 mLsamples=[1,1,1,2,2,2,3,3,3,4,4,4,5,5,5]number_sample=0

Loop(len(samples))Routine_call('Injection')While(CAVRO_XC.input(1))While_end()number_sample +=1Loop_end()

Routine_define('Injection')CAVRO_XC.initialize()

# PreconditioningCAVRO_XC.valve(7)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.aspirate_uL(200)CAVRO_XC.set_speed_uL_min(500)CAVRO_XC.valve(2)CAVRO_XC.empty()

CAVRO_XC.valve(4)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.aspirate_uL(500)CAVRO_XC.set_speed_uL_min(500)CAVRO_XC.valve(2)CAVRO_XC.empty()

CAVRO_XC.valve(1)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.fill()CAVRO_XC.valve(2)CAVRO_XC.set_speed_uL_min(500)CAVRO_XC.empty()

# Priming sampleAIM3200.position(samples[number_sample])CAVRO_XC.valve(5)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.aspirate_uL(200)CAVRO_XC.valve(9)CAVRO_XC.empty()

# Sample LoadingCAVRO_XC.valve(5)CAVRO_XC.set_speed_uL_s(500)

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CAVRO_XC.aspirate_uL(200)CAVRO_XC.set_speed_uL_min(200)CAVRO_XC.valve(2)CAVRO_XC.empty()

# DryingCAVRO_XC.valve(1)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.aspirate_uL(500)CAVRO_XC.valve(2)CAVRO_XC.set_speed_uL_min(200)CAVRO_XC.empty()

# Syringe cleaningLoop(2)CAVRO_XC.valve(7)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.fill()CAVRO_XC.valve(9)CAVRO_XC.empty()Loop_end()

Loop(2)CAVRO_XC.valve(4)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.fill()CAVRO_XC.valve(9)CAVRO_XC.empty()Loop_end()

# Rinsing of the monolithCAVRO_XC.valve(4)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.aspirate_uL(200)CAVRO_XC.set_speed_uL_min(200)CAVRO_XC.valve(2)CAVRO_XC.empty()

CAVRO_XC.valve(1)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.aspirate_uL(500)CAVRO_XC.valve(2)CAVRO_XC.set_speed_uL_min(200)CAVRO_XC.empty()

# ElutionCAVRO_XC.valve(7)CAVRO_XC.set_speed_uL_s(500)

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CAVRO_XC.aspirate_uL(50)CAVRO_XC.set_speed_uL_min(200)CAVRO_XC.valve(2)CAVRO_XC.empty()

# Heartcut parkingCAVRO_XC.valve(1)CAVRO_XC.set_speed_uL_s(500)CAVRO_XC.aspirate_uL(200)CAVRO_XC.valve(2)CAVRO_XC.set_speed_uL_min(200)CAVRO_XC.empty()

Routine_end()

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Table S2. Chromatographic operational conditions for HPLC separation and detection of target

anti-microbial species

HPLC-PDAMobile phase A 90% H2O, 10% ACNMobile phase B 100% ACN

Flow rate 0.6 mL min-1

Multilinear Gradient

0-2.2 min: 90% mobile phase A; 2.2-5.2 min, 55% mobile phase A; 5.2-6.2 min: keep 55% mobile phase A; 6.2-9.2 min: 100% mobile phase B; 9.2-9.3 min, 90% mobile phase A; 9.3-13 min: keep 90% mobile phase

AHPLC Column Onyx monolithic HD-C18 (100 × 4.6 mm)

Column temperature

30 ºC

Volume injection 50 µLDetection

Wavelength210 nm

Rotary valve (HPLC) in inject position (time)

10 s

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