ars.els-cdn.com · Web viewDendrimer-encapsulated gold nanoparticles were synthesized, according to Kim et al. (Kim et al., 2004), through reduction of dendrimer-encapsulated Au3+

SUPPORTING INFORMATION

An electrochemical immunosensor using gold nanoparticles-PAMAM-

nanostructured screen-printed carbon electrodes for tau protein

determination in plasma and brain tissues from Alzheimer patients

Claudia A Razzinoabζ Veroacutenica Serafiacutena ζ Maria Gamellaa ζ Mariacutea Pedreroa Ana Montero-

Callec Rodrigo Barderasc Miguel Calerod Anderson O Loboe Paloma Yaacutentildeez-Sedentildeoa

Susana Campuzanoa Joseacute M Pingarroacutena

ζThese authors contributed equally to this work

To whom correspondence should be addressed (susanacrquimucmes

pingarroquimucmes)

CONTENTS PAGES

MATERIALS AND METHODS S2-S6

Apparatus and electrodes S2-S3

Reagents and solutions S3-S4

Synthesis of 3D-Au-PAMAM S4

Immunosensor preparation S4-S5

Electrochemical measurements S5-S6

Analysis of real samples S6

RESULTS AND DISCUSSION S7-S18

Characterization of 3D-Au-PAMAM S7-S10

Fig S1 S7-S8

Fig S2 S9

Fig S3 S10

Optimization of experimental variables S10-S13

S1

Fig S4 S10

Table S1 S12

Fig S5 S13

Fig S6 S14

Table S2 S15

References S15-S16

MATERIALS AND METHODS

Apparatus and electrodes

Amperometric measurements were made with a CH Instruments potentiostat (model 812B

Austin TX) controlled by the CHI812B software Cyclic voltammetry (CV) and

electrochemical impedance spectroscopy (EIS) measurements were carried out using a FRA2

microAutolab Type III potentiostatgalvanostat (Metrohm Autolab BV The Netherlands)

controlled by the GPES amp FRA software (Eco Chemie BV The Netherlands) Screen-

printed carbon electrodes (SPCEs ref DRP-110 ϕ = 4 mm) and the connector cable (ref

DRP-CAC) were purchased from Metrohm DropSens SL (Spain) All the electrochemical

measurements were at room temperature

A vortex mixer (Velp Scientifica model Wizard IR Infrared) a pH-meter (Crison model

Basic 20+) a centrifuge (Med Instruments model MPW-65R) a thermomixer MT100

incubator shaker (Universal Labortechnik) and a magnetic stirrer (Metrohm model 728)

were also employed

The UV-Vis spectroscopy studies were carried out with UV-Vis spectrophotometers (Jasco

models V-630 and V-670) controlled by Spectra manager II software and the transmission

electron microscopy (TEM) and Energy-dispersive X-ray (EDX) characterization was

S2

performed using a transmission electron microscope (FEI TECNAI Gsup2F20 HRTEM)

operating at 120 and 200 kV

Reagents and solutions

All reagents were of the highest available analytical grade and used without further

purification Sodium nitrite (NaNO2) from Panreac 4-aminobenzoic acid (p-ABA) and

aminothiophenol (S-Phe) from Across and hydrogen tetrachloroaurate (III) trihydrate

(HAuCl43H2O) from Alfa Aesar were used PAMAM dendrimer ethylenediamine core

generation 40 solution (PAMAM) sodium borohydride (NaBH4) N-(3-dimethyl-

aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) N-hydroxysulfo-succinimide (Sulfo-NHS)

hydroquinone (HQ) Tweenreg 20 hydrogen peroxide (H2O2) (30 wv) human serum

albumin (HAS) hemoglobin (Hb) and human IgG were purchased from SigmandashAldrich

Bovine serum albumin (BSA) was purchased from Gerbu BlockerTM Casein in phosphate

buffered saline (PBS) (Ref 37528 casein blocking buffer CBB solution) and Piercetrade

Protein-Free PBS (Ref 37572 protein-free blocking buffer PFBB solution) solutions were

purchased from Thermo Scientific Recombinant human tau-441 (2N4R) (Ref 842501 tau)

anti-tau antibody (Ref 806503 used as capture antibody CAb) and HRP-labeled anti-tau

antibody (Ref 814306 used as detector antibody HRP-DAb) were purchased from

BioLegend Inc (San Diego CA)

The following buffer solutions were used phosphate buffer saline (PBS) consisting of 001

mol Lminus1 phosphate buffer solution containing 0137 mol Lminus1 NaCl and 00027 mol Lminus1 KCl pH

75 005 mol Lminus1 phosphate buffer (PB) pH 60 PBS supplemented with 005 (wv)

Tweenreg 20 (PBST) 25 mmol Lminus1 MES buffer pH 50 Other solutions used were 100 μmol

Lminus1 PAMAM 20 mmol Lminus1 HAuCl4 03 mol Lminus1 NaBH4 prepared in 03 mol Lminus1 NaOH All

S3

solutions were prepared with ultrapure water (182 MΩ cm) from a Milli-Qtrade Element A10

System

Procedures

Synthesis of 3D-Au-PAMAM

Dendrimer-encapsulated gold nanoparticles were synthesized according to Kim et al (Kim et

al 2004) through reduction of dendrimer-encapsulated Au3+ ions by H2 generated by sodium

borohydride solution Briefly 35 mL of a 20 mM HAuCl4 aqueous solution were added into

a beaker containing 5 mL of 10 microM PAMAM aqueous solution protected from light

Magnetic stirring was kept for 20 min Under magnetic stirring a 43-microL aliquot of a freshly

prepared 03 M NaBH4 basic solution was then added The resulting 3D-Au-PAMAM

suspension stable at least for 2 months was stored in a refrigerator and used after 24 h

without purification The synthetized 3D-Au-PAMAM nanocomposite was characterized by

UV-Vis spectroscopy Transmission Electron Microscopy (TEM) High Resolution

Transmission Microscopy (HRTEM) Energy Dispersive X-ray (EDX) and Cyclic

Voltammetry (CV)

Immunosensor preparation

The aryl diazonium salt was prepared by adding dropwise under constant magnetic stirring

2 mmol L-1 NaNO2 aqueous solution to 10 mg of p-ABA dissolved in 10 mL of 1 M HCl

cooled in an ice bath (38 μL of NaNO2 aqueous solution for each 200 μL of p-ABA acid

solution) The SPCEs were immersed in 1 mL of this resulting solution and ten successive

cyclic voltammetric scans were made between 00 and minus10 V (v = 200 mV sminus1) (Moreno-

Guzmaacuten et al 2012) The SPCEs functionalized by p-ABA electrografting (p-ABA-SPCEs)

were washed thoroughly with ultrapure water dried at room temperature and activated by

S4

coating the working electrode with a 10-microL drop of a EDCSulfo-NHS mixture solution (01

mol Lminus1 each and freshly prepared in 25 mmol Lminus1 MES buffer solution pH 50) and let to

stand for 30 min at room temperature in a humid chamber ndash this condition was used for all

incubation steps After rinsing with 25 mmol Lminus1 MES buffer solution pH 50 and drying at

room temperature 10 μL of the 3D-Au-PAMAM suspension were casted on the EDCSulfo-

NHS-activated p-ABA-SPCEs and let to react for 30 min Then the 3D-Au-PAMAM-p-

ABA-SPCEs were rinsed with ultrapure water dried at room temperature and incubated with

5 microL of a 05 (vv) GA solution (prepared in PBS) for 60 min After rinsing the GA-

activated-3D-Au-PAMAM-p-ABA-SPCEs with PBST a 5-microL aliquot of a 10 microg mLminus1 CAb

solution was added and let to react for 60 min The CAb-3D-Au-PAMAM-p-ABA-SPCEs

were rinsed with PBS and a blocking step with 10 microL of PFBB solution was carried out for 45

min Finally the modified electrodes were washed several times and kept at 4 ordmC until use

The sandwich immunoassay was carried out by incubating for 60 min the CAb-3D-Au-

PAMAM-p-ABA-SPCE with 5 microL of a mixture solution containing the tau standard (or the

sample to be analyzed) and 01 microg mLminus1 of HRP-DAb prepared in blocker casein solution

(CBB) The modified electrodes were washed with PBS solution and kept in a 10 microL-drop of

PBS pH 75 at room temperature until the electrochemical measurements were carried out

Electrochemical measurements

The stepwise preparation of the immunosensor was electrochemically characterized by CV

and EIS CV experiments were performed at 50 mV sminus1 over a potential range of either 00 to

+14 V or minus03 to +10 V in either 01 M H2SO4 or 01 mol Lminus1 KCl containing 50 mmol Lminus1

[Fe(CN)6]3minus4minus respectively The EIS measurements were carried out under open circuit

conditions in a frequency range of 100 kHz to 004 Hz with a sinusoidal signal of 10 mV

The frequency interval was divided into 50 logarithmically equidistant measure points

S5

Amperometric measurements were conducted in stirred solutions at minus020 V vs Ag pseudo-

reference electrodes after immersing the immunosensor into an electrochemical cell

containing 10 mL of 50 mmol Lminus1 PB solution pH 60 supplemented with 10 mmol Lminus1 HQ

(freshly prepared) After stabilization of the background current 50 μL of a 01 mol Lminus1 H2O2

fresh solution were added and the variation in the cathodic current was recorded until

reaching the steady-state current

All the error bars shown in the Figures were estimated as a triple of the standard deviation

(n=3)

Analysis of real samples

Plasma and brain tissue samples were obtained from the CIEN Foundationrsquos Tissue Bank

(BT-CIEN) According to the brain bankrsquos protocols neuropathological diagnosis and

classification of cases was performed on the basis of international consensus criteria (Thal et

al 2002 Mirra et al 1993) Written informed consent was obtained from all patients

Brain tissue protein extraction was performed as previously reported (Barderas et al 2012

Barderas et al 2013) Briefly tissue samples were cut in small pieces in dry ice and

mechanically disaggregated with 05 SDS in phosphate buffered saline (PBS) with a

protease inhibition cocktail (Sigma) and finally clarified by centrifugation at 10000 rpm

Brain tissue protein extracts were stored at ndash 80 ordmC until use Protein extracts quality was

assessed prior to be used in any experiment by Coomassie staining of SDS-PAGE 10 gels

RESULTS AND DISCUSSION

Characterization of 3D-Au-PAMAM

The synthesized 3D-Au-PAMAM nanocomposite was characterized by UVndashvis absorption

EDX TEM HRTEM and CV

S6

Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-

PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220

nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-

charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is

featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black

curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the

band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost

disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated

the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the

addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was

attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm

(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of

AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-

Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis

of the 3D-Au-PAMAM nanocomposite

S7

Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus

HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis

spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM

nanocomposite

Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension

The images reveal well separated Au nanoparticles due to the high charge density on the

PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The

AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al

2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent

nanoparticles agglomeration and the charged surface exerts only a slight influence over the

size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be

due to small particles that overlap in two-dimensional projection of images and appear as a

larger particle or even to real small agglomerations

A significant dependence of the nanocomposite structure on the dendrimer generation was

reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation

dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these

complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers

Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-

PAMAM and subsequently capped by other polymer units (Hofman et al 2011)

S8

10 nm 2 nm

a) b)

Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension

The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through

covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4

As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic

feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the

Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction

is observed within this material at +045 V

For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto

a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be

observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-

SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger

3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino

groups available for immobilization in the nanocomposite

S9

-02 00 02 04 06 08 10 12 14 16

-40-20

020406080

100120 3D-Au-PAMAM-S-Phe-SPCE

3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE

i

A

E V vs Ag pseudo-reference electrode

Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-

PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)

Optimization of experimental variables

00

10

20

30

40

50

i

A

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2

3

SB

(-) HQ (+) HQWithout 3D-Au-PAMAM

With 3D-Au-PAMAM

Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ

in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-

SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios

S10

Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its

incubation time the GA and CAb concentrations and the corresponding incubation times the

type and incubation time of blocking agent the number of steps involved in the immunoassay

the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were

checked The better ratio between the currents measured with the as prepared immunosensor

in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as

the selection criterion for each tested variable The tested ranges and the selected values are

summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting

Information It is important to note the lack of discrimination between the absence and the

presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In

addition a similar discrimination in the absence and in the presence of tau (SB ratio) was

found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein

with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in

the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-

Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied

potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those

optimized in previous works Moreover pH and temperature two key variables in the

functioning of the HRP enzyme and therefore of the developed immunosensor were selected

according to the available literature A pH value of 60 allows maximum enzyme activity

(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)

Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for

higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with

the aim to facilitate the implementation in POC devices we decided to develop the

methodology at room temperature

S11

Table S1

Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-

tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of

tau protein

Working variable Tested range Selected value

Volume of 3D-Au-PAMAM suspension microL 2540 10

Incubation time in 3D-Au-PAMAM suspension

min

15ndashon 30

GA concentration (vv) 0ndash25 05

Incubation time in GA solution min 30ndash120 60

CAb concentration microg mLminus1 0ndash25 10

Incubation time in CAb solution min 15ndashon 60

Incubation time in PFBB solution min 30ndash90 45

Number of steps 1ndash2 1

HRP-DAb concentration microg mLminus1 005ndash10 01

Incubation time in tauHRP-DAb mixture

solution min

15ndash120 60

on overnight

S12

0 5 10 250

20

30i A

CAb g mL-1

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2

3

SB

0

20

30

40

i

A

CAb incubation time min

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2

3

SB

005 01 025 05 10

20

30

40

i

A

Ab-HRP g mL-1

0 ng mL-1 TAU 5 ng mL-1 TAU

0

1

2

3

4

SB

0

2

3

4

5

6i A

0 ng mL-1 TAU 5 ng mL-1 TAU

00

05

10

15

20 SB0

20

30

40

50

60

i

A

0 ng mL-1 tau 5 ng mL-1 tau

00

05

10

15

20

SB

1 STEP 2 STEPS0

20

30

40

-i A

0 ng mL-1 TAU 5 ng mL-1 TAU

00

05

10

15

20

SB

30 45 60 90 1200

20

30i

A

Glutaraldehyde incubation time min

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2

3

SB

25 5 10 40 10 (dry)0

20

30

40

i

A

Volumme3D-Au-PAMAM microL

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2 SB

15 30 45 60 90 1200

10

20

30

40

0 ng mL-1 TAU 5 ng mL-1 TAU

i

A

HRP-DAb incubation time min

0

1

2

3

SB

a)

0 025 05 10 250

20

30i A

Glutaraldehyde

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2

3

SB

0

20

30

40

50

i

A

3D-Au-PAMAM incubation time min

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2

3

SB

c) d)

f) g) h)

30 45 60 900

20

30i

A

Blocking agent incubation time min

0 ng mL-1 tau 5 ng mL-1 tau

0

1

2

3

SB

i) j) k)

b)

e)

Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates

S13

0 3 7 9 270

20

30

40

50

0 ng mL-1 tau 25 ng mL-1 tau

i

A

Days

0

1

2

3

SB

Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)

Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1

tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of

the standard deviation of three replicates

S14

Table S2

Determination of tau in spiked plasma and brain tissue extract samples

Sample[tau]spiked

pg mLminus1

[tau]found

pg mLminus1 Recovery

Brain tissue

protein extracts

Healthy

individual

200 261 plusmn 3 125 plusmn 2

600 613 plusmn 45 101 plusmn 8

AD

patient

Broak IV

200 229 plusmn 10 114 plusmn 6

600 562 plusmn 4 94 plusmn 5

PlasmaHealthy

individual20 204 plusmn 04 102 plusmn 4

Mean values plusmn tsradicn n=3 α=005

Value obtained by subtracting the determined endogenous content shown in Table 1 from

the total found content

References

Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM

Alamery SF 2019 Saudi J Biol Sci 26 301ndash307

Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I

Whetten RL 1997 J Phys Chem B 101 3706ndash3712

Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez

R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55

Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A

Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938

Camarada MB 2017 J Phys Chem A 121 8124minus8135

S15

Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP

Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88

Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P

Pingarroacuten JM 2010 Biosens Bioelectron 26 517522

Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608

Hamelin A 1996 J Electroanal Chem 407 1ndash11

Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir

27 759ndash6767

Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172

Li D Li J 2003 Chem Phys Lett 372 668ndash673

Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136

4500ndash4506

Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144

Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P

Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86

Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35

Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr

Microbiol App Sci 4 367ndash375

Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800

Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004

Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13

29212933

S16