Covalent functionalisation controlled by molecular design for the aptameric recognition of serotonin in graphene-based field-effect transistors

In the last decade, solution-gated graphene field effect transistors (GFETs) showed their versatility in the development of a miniaturized multiplexed platform for electrophysiological recordings and sensing. Due to their working mechanism, the surface functionalisation and immobilisation of receptors are pivotal to ensure the proper functioning of devices. Herein, we present a controlled covalent functionalisation strategy based on molecular design and electrochemical triggering, which provide a monolayer-like functionalisation of micro-GFET arrays retaining the electronic properties of graphenes. The functionalisation layer as a receptor was then employed as the linker for serotonin aptamer conjugation. The micro-GFET arrays display sensitivity toward the target analyte in the micromolar range in a physiological buffer (PBS 10 mM). The sensor allows the in-flow real-time monitoring of serotonin transient concentrations with fast and reversible responses.


Material and methods
All the chemicals for the synthesis of the mMAL-DS and MAL-DS were purchased from Sigma-Aldrich, Germany.Phosphate buffer saline tablets (PBS), potassium nitrate (KNO3, ≥99.0%), tris(2carboxyethyl)phosphine hydrochloride (TCEP, ≥98.0%), serotonin hydrochloride (≥98%) and 2mercapto ethanol (55 mM in PBS) were also from Sigma-Aldrich.Zeba™ Spin Desalting Columns, 7 K MWCO, 0.5 mL were purchased from Thermofisher, France.The 5'-thiol modified aptamer (5'-CGA CTG GTA GGC AGA TAG GGG AAG CTG ATT CGA TGC GTG GGT CG-3') and scrambled sequence (5'-CCC GGG AAT TCC GGA ATT GGG GCA ATT GAT GAG GGG GTC ATG GG-3') were purchased from Microsynth, Switzerland.Chemical vapour deposited graphene (CVDg) on copper was purchased from Graphenea, Spain.AFM micrographs were registered using a JPK BioAFM microscope (Bruker Nano GmbH), employing a tapping mode tip with the frequency of 320 Hz (Bruker TESPA-V2).Shaving experiments were performed on a Bruker Multimode 8 microscope in Contact mode scanning several times an area of 10 µm x4 µm and applying a force of ≈70 nN at a scan rate of 2Hz.The probe used was the ScanAsyst-Air silicon tip (Bruker).The shaved area was characterized again in PeakForce QNM mode with a scan angle of 90o to avoid artifacts do to piled material.AFM images were processed using WSvM5.0 and Nanoscope Analysis 2.0 software.XPS measurements were performed in a SPECS Sage HR 100 spectrometer using a non-monochromatic X-ray source of Mg with a Ka line of 1253.6 eV.An electron flood gun was used to avoid sample charging.XPS data fitting was performed using Casa XPS software (Version 2.3.16PR 1.6).Raman spectra were recorded using a Renishaw Invia Raman spectrometer (lex = 532 nm).Each spectrum is the average of at least 300 spectra recorded in different spots of the sample with 1 s of integration time at a laser power of 1.29 mW.Data were processed using Ranishaw WiRE 4 software.All NMR data were collected on a Bruker AVANCE III NMR spectrometer (11.7 T, 500 MHz for 1H).The spectra were processed with MestReNova software version 7.1.1-9649.Electrochemical characterizations were performed through CV and at constant potential with an electrochemical workstation Autolab dropwise to the SiO 2 /CVDg soaked in Milli Q water (10 mL) using a syringe pump system (flow rate 2.5 mL min -1 ).The substrate was then rinsed with abundant Milli Q water and ethanol and dried with N 2 flow.

GFET fabrication
The same fabrication process was used to manufacture the macro-GFET and the 14-channel single-address GFET and the 48-channel micro-GFET.A conventional lift-off process using the image reversal photoresist AZ5214E (Clariant, Germany) is followed in four-inch silicon wafers covered by 2 µm thick thermal oxide.The bottom metal layer is an e-beam evaporated Ti/Au, 10/100 nm in thickness.After graphene transfer, the graphene GFET active areas were defined by means of an oxygen-based reactive ion etching using a patterned HiPR 6512 resist mask which is subsequently removed in acetone.The patterning process of the top metal layer is similar to the bottom metal contact patterning, but the metal stack consisted in a Ni/Au, 20/200 nm thick film.
Finally, SU-8 negative photoresist (SU-8 2005, MicroChem, USA) is used to passivate the metal leads while defining the graphene channel and metal contacts openings.

GFET functionalization
The electrochemical functionalization was performed using the macro/micro-GFET as WE in a tree-electrode configuration using Ag/AgCl (KCl 3M) as a RE and platinum plate as a CE.The electrodes were soaked in a solution mMAL-DS in 50% ethanol with 0.1 M KNO 3 (5 mM) as support electrolyte.The functionalization was produced by performing a CA at -0.5 V vs Ag/AgCl (KCl 3M) for 100s (unless stated otherwise).The GFET was then rinsed with ethanol and Milli Q water and dried.The electrografting performed by CV (E= -0.7: 0 V vs Ag/AgCl (KCl 3M), 3 or 4 scans, 0.1 V s -1 scan rate) was performed with the same experimental set up and mMAL-DS concentration.
Ferrocene-SH conjugation 50 µL of tris(2-carboxyethyl)phosphine (TCEP) solution (10 mM in PBS 10 mM) was freshly prepared and mixed with of PBS (10 mM, 100 µL) and Ferrocene-C 6 H 12 -SH solution (2 mM in EtOH, 50 µL).The mixture was shaken for 30 minutes and then the solution (100 µL) was incubated overnight over the device.The device was then washed with H 2 O and EtOH before further analysis.

S5
Stem-loop aptamer conjugation The as received aptamer was diluted up to 100 µM in 10 mM PBS containing 50-fold excess of TCEP and incubated for 1 hour.The solution was purified using a ZEPA® desalting column (MWCO 7kDa) before aliquoting and storing at -20°C until further use.
Prior the surface conjugation, the aptamer was refolded by treatment at 90 °C for 10 minutes followed by cooling at r.t..The solution was then diluted to 1 µM in 10mM PBS and incubated on the device overnight.The device was then delicately rinsed with Milli Q water and gently dried.The surface groups were subsequently blocked by incubating a solution 2-mercapto ethanol (1 mM in 10 mM PBS) for 20 minutes.The device was washed and dried prior to further characterization.

Electrical characterization of GFET
The setup to perform the measurements is composed by a Printed Circuit Board (PCB) connected to a Data Acquisition Card (DAQ Card), which digitalizes and transmits to a computer the analogue signal and allow the measurement of 24 transistors of each probe and the Direct Current (DC) source that polarize the operational amplifiers from the PCB.The transfer curves were recorded in 10mM PBS sweeping the V GS between -0.1 and 0.4 V using an Ag/AgCl (KCl 3M) RE as the gate.The device was connected to the electrical setup through a PMMA cell equipped with gold arrays in touch with the gold contacts.The V DS was maintained fixed to 0.05 V vs Ag/AgCl (KCl 3M) for all the experiments.
Serotonin monitoring A PMMA flow cell connected to a syringe pump through a 6-port valve.was connected to the Ag/AgCl (KCl 3M) gate by the outlet tube ending in a 10 mM PBS, i.e. PBS 1x, reservoir.Here, the V GS was fixed at 0 V vs Ag/AgCl (KCl 3M).During the experiment the flow rate was kept at 100 µL min -1 during the injection.Then it was lowered to 10 µL min -1 once the solution reached the cell and maintained for 8 min, before injecting the following solution.The data were analysed via a Python 3 scripts.

Synthesis of mMAL-DS
Scheme 1. Synthetic route for mMAL-DS (1i).2',6'-dimethyl-4-nitro-p-toluensulfanilide (1c) was synthetized according to a modified literature procedure.2',6'-dimethyl-p-toluensulfanilide (67.4 mmol, 18.6 g) was dissolved in ethyl acetate (50 mL) at reflux.99% HNO 3 (74.1 mmol, 3.0 mL) was dissolved in ethyl acetate (20 mL) were added dropwise for 10 minutes and the reflux was continued for 2h30'.The reaction mixture was poured on crushed ice, the precipitate was filtered and washed with water.The solid was redissolved in dichloromethane, activated charcoal was added and the mixture was stirred for 20 minutes and filtered through celite.The solvent was evaporated, and the product was finally triturated with methanol.The compound 1c (36.4 mmol, 11.91 g) was obtained as a white solid in 54% yield.
The solvent was evaporated under reduced pressure and the product was purified by column chromatography in hexane / AcOEt (80:20 → 70:30).The product was decanted in hexane (20 mL), the coloured supernatant was removed, and the precipitate was washed with hexane (2 x 5 mL).
The compound (8.8 mmol, 1.56 g) was obtained as a pale violet solid in 99% yield.

GFET fabrication
The fabrication process was employed for the fabrication of macro-GFET, 14 channels singleaddressed GFET and 48-channels micro-GFET.A conventional lift-off process using the image reversal photoresist AZ5214E (Clariant, Germany) is followed in four-inch silicon wafers covered by 2 μm thick thermal oxide.The bottom metal layer is an e-beam evaporated Ti/Au, 10/100 nm in thickness.After graphene transfer, the graphene GFET active areas were defined by means of an oxygen-based reactive ion etching using a patterned HiPR 6512 resist mask which is subsequently removed in acetone.The patterning process of the top metal layer is similar to the bottom metal contact patterning, but the metal stack consisted in a Ni/Au, 20/200 nm thick film.Finally, SU-8 negative photoresist (SU-8 2005, MicroChem, USA) is used to passivate the metal leads while defining the graphene channel and metal contacts openings.
Prior to CVD process, copper foils were cut in 6 x 5 cm 2 samples and sequentially cleaned in acetic acid and acetone, and finally rinsed in isopropyl alcohol (IPA).Graphene growth process was composed by two steps; i) 10 minutes Cu annealing at 1000 °C, with flow of 400 standard cubic centimetres per minute (sccm) of H 2 , 600 sccm of Ar and 150 sccm of N 2 , followed by ii) 10 minutes graphene growth at 970 °C using methane as carbon precursor (10/100 sccm CH 4 / H 2 ratio).A 700 nm thick sacrificial layer of polymethyl methacrylate (PMMA, 7% 950k MW PMMA dissolved in anisole, provided by Micro Resist technology GmbH, DE) was deposited by spin-coating above graphene.CVD graphene was delaminated from the Cu foil by the electrochemical method.42Before the transfer process, supporting substrates were activated by UVO cleaner for 5 minutes.After transfer, samples underwent to a 40 min temperature ramp from r.t. to 180°C and a final bake at 180 °C for 2 minutes.After cooling to r.t., the PMMA sacrificial layer was removed by immersing samples in acetone and then in isopropanol, for 30 minutes in each solvent.
Graphene was grown by Chemical Vapor Deposition (CVD) method.Black magic Pro 4" CVD Aixtron Reactor and 25 µm thick, 99.8% metal basis copper foil S4 provided by Alfa Aesar have been employed.