Tailoring Catalytic Activity of Pt Nanoparticles Encapsulated Inside

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Tailoring Catalytic Activity of Pt Nanoparticles Encapsulated Inside Dendrimers by Tuning Nanoparticle Sizes with Sub-nanometer Accuracy for Sensitive Chemiluminescence-based Analyses Hyojung Lim, Youngwon Ju, and Joohoon Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00073 • Publication Date (Web): 01 Apr 2016 Downloaded from http://pubs.acs.org on April 2, 2016

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Analytical Chemistry

Tailoring Catalytic Activity of Pt Nanoparticles Encapsulated Inside

Dendrimers

nanometer

Accuracy

by

Tuning for

Nanoparticle

Sensitive

Sizes

with

Sub-

Chemiluminescence-based

Analyses

Hyojung Lim,a,† Youngwon Ju,a,† and Joohoon Kima,b,*

a

Department

Science

and

of

Chemistry,

Technology,

b

KHU-KIST

Kyung

Hee

Department University,

of

Converging

Seoul

130-701,

Republic of Korea

*To whom correspondence should be addressed. E-mail: [email protected]; Voice: 82-2-961-9384; Fax: 82-2-9610443

†Equally contributed to this research

1 Environment ACS Paragon Plus


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Abstract Here, we report the size-dependent catalysis of Pt dendrimerencapsulated nanoparticles (DENs) having well-defined sizes over the range of 1 ~ 3 nm with sub-nanometer accuracy for the highly enhanced chemiluminescence of the luminol/H2O2 system. This sizedependent

catalysis

is

ascribed

to

the

differences

in

the

chemical states of the Pt DENs as well as in their surface areas depending on their sizes. Facile and versatile applications of the Pt DENs in diverse oxidase-based assays are demonstrated as efficient

catalysts

for

sensitive

chemiluminescence-based

analyses.


Page 3 of 41

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Introduction Controlling nanoparticle sizes has become increasingly important for

tuning

reactions

nanoparticle

including

catalysis

in

a

chemiluminescence.1-3

variety

of

chemical

Chemiluminescence

has

been a versatile and sensitive tool for analytical applications in diverse fields including molecular biology, biotechnology, medical science, pharmacology, and chemistry.4-7 This is mainly because

of

the

chemiluminescence which

provides

background,

unique

originating

advantages

simple

light from

over

emission chemical

process

redox

photoluminescence

instrumentation,

and

of

reactions,

such

as

robustness.8

low The

luminol/H2O2 system has been one of the most frequently used chemiluminescence systems since the chemiluminescence phenomenon of

luminol

was

first

reported

in

1928.9-11

In

particular,

substantial efforts have been made in the amplified generation of chemiluminescence of the luminol/H2O2 system to facilitate its further

use

in

analytical

applications

with

improved

sensitivity. In the search for highly efficient catalysts for the amplified chemiluminescence, unique nanoparticles as well as conventional catalysts such as enzymes, metal ions, and metal complexes have often been explored.12-15 For example, Au and Pt nanoparticles

have

been

employed

to

catalyze

the

chemiluminescence of luminol/H2O2, allowing amplified analysis of small organic compounds, aptamer-protein binding interactions,



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DNA.16-18

and

Since

size-dependent

Page 4 of 41

catalytic

properties

of

nanoparticles have been the subject of extensive investigations in catalysis studies, it is also of particular interest to study the

size-dependent

chemiluminescence.

catalysis

Indeed,

of

recent

nanoparticles

studies

indicated

in

that

the

size of nanoparticles affects the catalytic activity for the amplified generation of the chemiluminescence of luminol/H2O2, 16,17

which suggests further investigation of catalytic properties

of

nanoparticles

of

different

particle

sizes

to

be

highly

desired for the rational design of new efficient catalysts in chemiluminescence.

Especially,

it

would

be

of

interest

to

investigate the catalytic activity of small nanoparticles in the range of sizes less than 3 nm since such small catalysts have been rarely explored for the enhanced chemiluminescence in spite of the unique features of very small nanoparticles in this range of small sizes.19 In

this

dependent

context,

catalytic

we

here

activity

report of

the

Pt

significant

size-

dendrimer-encapsulated

nanoparticles (DENs) having well-defined sizes over the range of 1

~

3

nm

with

sub-nanometer

accuracy

for

the

enhanced

chemiluminescence of the luminol/H2O2 system. We also demonstrate the versatile applications of the Pt DENs in diverse oxidasebased

assays

as

efficient


analyses.

catalysts Specifically,


for

sensitive

we

synthesized

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Pt DENs using amine-terminated nth generation polyamidoamine (GnNH2 PAMAM, n = 6 and 4) dendrimers and three different Pt2+/Gn-NH2 ratios (i.e. 55, 147, and 200), which we denote as G6-NH2(Pt55), G6-NH2(Pt147),

G6-NH2(Pt200),

and

G4-NH2(Pt55),

respectively.

The

synthesized Pt DENs showed different sizes, which can be readily controlled by adjusting the Pt2+/Gn-NH2 ratios, but were fairly uniform and monodispersed in size with sub-nanometer accuracy. The Pt DENs exhibited significant catalytic activity for the generation of reactive oxygen species such as hydroxyl radical (OH·), superoxide radical anion (O2·–), and singlet oxygen (1O2), leading to the highly enhanced chemiluminescence of luminol/H2O2. Especially, the G6-NH2(Pt200) DENs displayed ca. 12-fold increase in the chemiluminescence emission compared to that obtained in the

absence

of

the

Pt

DENs

while

maintaining

its

catalytic

activity even with harsh thermal perturbation. More importantly, the catalysis of Pt DENs in the generation of chemiluminescence is quite sensitive to the sizes of Pt DENs, even with the subnanometer

changes

in

size,

which

is

ascribed

to

different

extents of fully reduced Pt DENs as well as different surface areas.

The

catalysis

chemiluminescence

was

of also

Pt found

DENs to

for be

the

dependent

enhanced on

the

generation of dendrimers (i.e. G6-NH2 or G4-NH2). Finally, we demonstrated the analytical versatility of the Pt DEN-catalyzed generation of chemiluminescence in oxidase-based analyses toward



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various

oxidase

substrates

Page 6 of 41

including

choline,

glucose,

and

cholesterol.

Experimental Section Chemicals and materials. Amine-terminated nth generation PAMAM dendrimers (Gn-NH2, n = 6 or 4), K2PtCl4, NaBH4, Na2CO3, NaHCO3, CH3COONa, (CH3CO)2O, Na2HPO4 · 7H2O, NaH2PO4 · 2H2O, NaOH, TritonTM X-100, luminol (97%), H2O2 (30 wt% in water), NaN3, thiourea, ascorbic

acid,

sodium

nitrotetrazolium

blue

piperidone

(TEMP),

aspergillus

niger),

alcaligenes

sp.),

salicylate, chloride,

glucose

chloride,

cholesterol,

benzoate,

2,2,6,6,-tetramethyl-4-

D-(+)-glucose, choline

sodium

oxidase

choline

cholesterol

(from

oxidase

(from

oxidase

(from

Streptomyces sp.), and cellulose sacks (MW cutoff of 12,000) were

purchased

alcohol

and

HCl

from

Sigma-Aldrich,

were

obtained

Inc.

from

(U.S.A.).

Dae-Jung,

Isopropyl

Inc.

(Korea).

Deionized (DI) 18 MΩ∙cm water was used in the preparation of aqueous solutions (Ultra370, Younglin Co., Korea). Characterization.

Transmission

electron

microscope

(TEM)

images were obtained using a JEM-3010 instrument (JEOL, U.S.A) operating at 300 kV. TEM samples were prepared by placing a drop of aqueous DEN solution on 400 mesh carbon-coated copper grids (Ted Pella Inc., U.S.A.) and allowing the grids to dry in air. UV-visible

absorption

spectra

were

obtained


using

an

Agilent

Page 7 of 41

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8453 UV-visible spectrometer (Agilent Tech., U.S.A.) with quartz cells (optical path length: 2 mm). DI water was used as blank solutions

for

the

UV-visible

absorption

spectra.

X-ray

photoelectron spectroscopy (XPS) spectra were acquired with a KAlpha

X-ray

photoelectron

spectrometer

(Thermo

Scientific,

U.S.A.) using Al Kα radiation (hν = 1486.6 eV). Electron spin resonance (ESR) spectra were collected using a Bruker EMXplus spectrometer (Bruker, U.S.A.). Synthesis of Pt DENs. Pt DENs were synthesized in a similar way to that previously reported by ourselves and other groups.20-22 Briefly, 55, 147, or 200 molar equivalent of 200 mM K2PtCl4 in water was added to a 10 µM Gn-NH2 (n = 6 and 4) PAMAM dendrimer aqueous

solution.

The

pH

value

of

the

mixture

solution

was

adjusted to 5 using 2 M aqueous HCl. The mixture solution was then stirred for 76 h to ensure complete complexation of the Pt2+ precursor ions (unless indicated otherwise, Pt2+ ions are used to represent all possible complex ions into the dendrimers)23 in the interior amines of the dendrimers. This results in the formation of the Pt2+ ions/dendrimer complexes, which we denote as GnNH2(Pt2+)m (n = 6 and 4, m = 55, 147, and 200). Next, a 20-fold excess of NaBH4 was added to the Gn-NH2(Pt2+)m complex solution under vigorous stirring. This mixture solution was kept in a closed vial for 24 h after adjusting the pH of the mixture below 7 for the reduction of the complexed Pt ions to Pt nanoparticles



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Page 8 of 41

inside the dendrimers. Finally, the resulting Pt DEN solution was dialyzed for 24 h using a cellulose dialysis sack to remove impurities.

The

actual

concentration

of

the

dialyzed

Pt

DEN

solution was also confirmed using the Agilent 8453 UV-visible spectrometer. Chemiluminescence

measurements.

All

chemiluminescence

measurements were carried out using a home-made cell (internal volume: 5 mL) with a quartz window connected to the slit of a monochromator

(Acton

U.S.A.)

equipped

(PIXIS

100

B,

photomultiplier Instruments,

Standard

with

a

U.S.A.).

device

Instruments,

(PMT) In

Princeton

charge-coupled

Princeton tube

SP2150,

detector

a

typical

Instruments, (CCD)

U.S.A.)

(PD-471,

measurement,

camera or

a

Princeton 50.5

µL

of

aqueous H2O2 solutions with different concentrations were added to the home-made cell containing luminol and Pt DEN in a 100 mM carbonate luminol

buffer and

Pt

Chemiluminescence

(pH

9.5).

DEN

were

spectra

The 5

mM

were

final and

concentrations 500

then

nM,

obtained

of

the

respectively. using

the

monochromator equipped with the CCD camera during the initial 10 s upon the addition of H2O2. 50 mM stock solution of luminol was prepared by dissolving luminol in 0.1 M NaOH aqueous solution, and stored under dark in a refrigerator. Working solutions of H2O2 were freshly prepared from 30 wt% H2O2.


Page 9 of 41

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Oxidase-based

assays

cholesterol,

and

performed

using

by

for

the

glucose.

The

three

different

detection

of

oxidase-based types

of

choline,

assays

were

oxidases

(i.e.

choline oxidase, cholesterol oxidase, and glucose oxidase) for the

detection

cholesterol,

of

the

and

corresponding

glucose,

substrates

respectively)

of

(i.e. the

choline, oxidases.

Specifically, the oxidase-based assays were carried out in 2 mL of the reaction buffer containing 0.05 mg/mL of each oxidase and various

concentrations

reaction

buffers

used

of for

the the

corresponding choline

substrate.

oxidase-based,

The

glucose

oxidase-based, and cholesterol oxidase-based assays were 100 mM phosphate buffer (pH 8.0), 100 mM sodium acetate buffer (pH 5.5), and 100 mM phosphate buffer (pH 7.0), respectively. The reaction mixtures were incubated at 37 °C for 30 min. 500.0 µL of each of the resulting mixtures was then added to the home-made cell containing luminol and Pt DEN in a 100 mM carbonate buffer (pH

9.5)

for

the

chemiluminescence

measurements

as

descried

earlier.

Results and Discussion Synthesis of Pt DENs having well-defined sizes over the range of 1

~

3

nm.

We

synthesized

four

different

Pt

DENs

(i.e.

G6-

NH2(Pt55), G6-NH2(Pt147), G6-NH2(Pt200), and G4-NH2(Pt55)) based on the

previously reported method (see the Experimental Section



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Page 10 of 41

for details). The synthetic method consists of two main steps: complexation of Pt2+ ions into the interior amines of dendrimers and subsequent chemical reduction of the complex Pt2+ ions inside the dendrimers.21,22 The four Pt DENs were synthesized using two different generation types of dendrimers (i.e. G6-NH2 and G4-NH2) and three different Pt2+/Gn-NH2 ratios (i.e. 55, 147, and 200). Note that the 4th generation dendrimer G4-NH2 was used only for the synthesis of G4-NH2(Pt55), which is the Pt DEN with a Pt2+/G4NH2 ratio of 55, since the G4-NH2 has only 62 interior tertiary amines, which is insufficient to be coordinated with all of the Pt2+ ions with high Pt2+/G4-NH2 ratios,23,24 which are 147 and 200 in the

present

synthesized

study. Pt

DENs.

Figure

1

The

TEM

shows images

TEM

images

indicate

of that

the the

asPt

nanoparticles are rarely aggregated, roughly spherical-shaped, and fairly monodispersed in size, suggesting stabilization of the

nanoparticles

via

their

encapsulation

inside

the

dendrimers.22,25 The complete size distribution histograms of the four Pt DENs, obtained from the TEM measurements, are provided in Figure S1 (Supporting Information). The measured diameters of the Pt nanoparticles for G6-NH2(Pt55), G6-NH2(Pt147), G6-NH2(Pt200), and G4-NH2(Pt55) are 1.2 ± 0.2 nm, 1.6 ± 0.2 nm, 1.8 ± 0.2 nm, and 1.2 ± 0.3 nm, respectively. The average diameters are very close to the theoretical values of 1.16 nm, 1.62 nm, 1.79 nm, and 1.16 nm, respectively, which are calculated by assuming a spherical


Page 11 of 41

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geometry of the nanoparticles containing 55, 147, or 200 metal atoms.26 These TEM results confirm that the four different Pt DENs have fairly well-defined uniform sizes with sub-nanometer accuracy in this range of sizes less than 3 nm. Furthermore, they suggest that the sizes of synthesized Pt DENs are readily controllable synthetic

by

adjusting

procedure.

The

Pt2+/Gn-NH2

the

UV-visible

ratios

spectroscopy

during

the

measurements

further verified the controllable synthesis of the Pt DENs with different sizes depending on the synthetic condition, i.e. the Pt2+/Gn-NH2 ratios (Supporting Information, Figure S2). Figure S2 shows the UV-visible absorption spectra of the four Pt DENs, which

reveal

wavelengths absorption

broad higher

at

λ

and than

≥

300

featureless 300 nm

nm

(λ

decreasing

absorbance

≥

300

nm).

The

indicates

the

formation

at

unique of

Pt

nanoparticles.23,27 The higher absorbance at λ ≥ 300 nm in the UVvisible spectra of the Pt DENs with higher Pt2+/Gn-NH2 ratios also suggests the increase in the sizes of the Pt nanoparticles as the Pt2+/Gn-NH2 ratio increases, which is in good agreement with the TEM results discussed earlier. It is also worth noting that the UV-visible spectra of G6-NH2(Pt55) and G4-NH2(Pt55) match each other, especially at λ ≥ 300 nm, indicating the different Pt DENs have the same size (ca. 1.2 nm as measured by the TEM), in spite of the different generation types of the dendrimers used for their synthesis, since their Pt2+/Gn-NH2 ratios are all 55.



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Catalytic

properties

chemiluminescence the

four

of

different

of

Pt

luminol/H2O2 synthesized

Page 12 of 41

DENs

system. Pt

DENs,

for

After we

enhanced

characterizing

investigated

the

catalytic features of the Pt DENs for enhanced chemiluminescence of the luminol/H2O2 system. Figure 2 shows the chemiluminescence spectra of the luminol/H2O2 system in either the presence or absence of the Pt DENs. In the presence of the Pt DENs, the spectra

displayed

significantly

higher

chemiluminescence

intensity than that obtained in the absence of the Pt DENs. Especially, the use of G6-NH2(Pt200) revealed ca. 12 times larger chemiluminescence emission ((i) in Figure 2) than the emission obtained in the absence of the Pt DENs ((vi) in Figure 2). The chemiluminescence

emission

is

chemiluminescence

intensity

chemiluminescence

spectra

defined

over

the

obtained

as

the

integrated

wavelength.

with

at

Multiple

least

three

independent G6-NH2(Pt200) DENs indicate (12 ± 3)-fold enhancement in

the

without

chemiluminescence the

Pt

DENs.

emission

However,

the

compared addition

to

the

of

emission

only

G6-NH2

dendrimers (without the encapsulated Pt nanoparticles) resulted in no significant increase in the emission ((vii) in Figure 2), which indicates that the observed catalytic chemiluminescence is attributed

to

the

dendrimer,

not

to

Pt the

nanoparticle dendrimer

encapsulated

itself.

We

also

inside

the

observed

a

negligible change in the chemiluminescence in the presence of


Page 13 of 41

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G4-NH2

dendrimers,

as

for

the

G6-NH2

(Supporting

Information,

Figure S3). As an additional control experiment, we added Pt2+ ion-dendrimer complexes with a Pt2+/G6-NH2 ratio of 200 (i.e. G6NH2(Pt2+)200)

to

the

luminol

reaction

solution

containing

H 2 O2

instead of the G6-NH2(Pt200) DENs. Interestingly, we observed a small

increase

addition

of

in

the

Pt2+

the

chemiluminescence

ion-dendrimer

intensity

complexes

to

upon

the

the

luminol

reaction solution ((v) in Figure 2), indicating slight catalytic activity

of

Pt2+

the

chemiluminescence

of

the

ion-dendrimer luminol/H2O2

complexes

system.

for

However,

the it

is

worth noting that the catalytic activity of the complexes is much smaller than that of the G6-NH2(Pt200) DENs. These results clearly confirm that the observed catalytic activity for the enhanced chemiluminescence of the luminol/H2O2 system originates from the Pt nanoparticles encapsulated inside the dendrimers. The chemiluminescence spectra of the luminol/H2O2 shown in Figure

2

also

demonstrate

two

additional

important

points.

First, the chemiluminescence spectra display maximum intensities at the same wavelength (ca. 440 nm), corresponding to the light emission

of

excited

3-aminophthalate

anions,28-30

in

both

the

presence and absence of the Pt DENs. This indicates that the chemiluminescence

emission

in

the

presence

of

the

Pt

DENs

originates from the same emitting species (i.e. the excited 3aminophthalate anions) as that in the absence of the Pt DENs.



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Second,

the

chemiluminescence

Page 14 of 41

spectra

show

different

maximum

intensities, in spite of being at the same wavelength as that discussed above, depending on the types of Pt DENs present in the luminol/H2O2 reaction solutions, which indicates different catalytic

activities

of

the

Pt

DENs

for

the

enhanced

chemiluminescence of luminol/H2O2. The catalysis of the Pt DENs in

the

chemiluminescence

of

luminol/H2O2

is

dependent

on

the

generation numbers of the dendrimers and the size of the Pt nanoparticles.

Specifically,

the

use

of

G4-NH2(Pt55)

DENs

exhibited 44% larger chemiluminescence emission ((iv) in Figure 2) than that for the G6-NH2(Pt55) DENs ((iii) in Figure 2), even though nominally the same Pt nanoparticles (i.e. Pt55 having the same average size of 1.2 nm) were encapsulated in the dendrimers of different generations (i.e. G4-NH2 and G6-NH2, respectively). This suggests that the catalytic activity of the Pt DENs can be controlled by using dendrimers of different generations. Since the lower generation dendrimers are more porous and thus more likely interior

to

take

the

catalytic

chemiluminescence

Pt

nanoparticles,

substrates the

higher

into

the

catalytic

activity of the G4-NH2(Pt55) DENs can be ascribed to the lower generation of dendrimers than that of the G6-NH2(Pt55) DENs.31,32 In addition, we found that the chemiluminescence emission increases significantly

as

the

encapsulated

inside

particle the

size

G6-NH2

of

the

dendrimers


Pt

nanoparticles

increases;

the

Page 15 of 41

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chemiluminescence emissions for the G6-NH2(Pt200) and G6-NH2(Pt147) DENs

are

311%

and

203%

(((i)

and

(ii)

in

Figure

2),

respectively, of that for the G6-NH2(Pt55) DENs ((iii) in Figure 2).

We

also

observed

the

same

trend

in

the

size-dependent

catalysis of the Pt DENs for the enhanced chemiluminescence of luminol/H2O2 luminol

when

upon

the

intensity-time different

monitoring addition

profiles,

sizes

in

the

dynamic

of

H 2 O2 ,

the

(Supporting

chemiluminescence

i.e.

presence

chemiluminescence

of

Information,

of

the

Pt

Figure

DENs

S4).

of

These

different catalyses of the Pt DENs depending on the dendrimers of different generations and the encapsulated Pt nanoparticles of different sizes were also observed in the chemiluminescence of luminol with different concentrations of H2O2 as shown in Figure 3a. Compared to the chemiluminescence obtained in the absence

of

the

Pt

DENs,

the

chemiluminescence

emissions

of

luminol, increasing linearly according to the concentrations of H2O2, were highly enhanced when using the Pt DENs. Importantly, the

catalysis

of

chemiluminescence generation encapsulated

number Pt

the

of

the

of

Pt

DENs

in

luminol/H2O2

the

system

dendrimers

nanoparticles

the

and

generation increases

the

decreases

size

and

of

as

the

of

the

increases,

respectively. This is an interesting finding since it indicates that

the

catalysis

of

the

Pt

DENs

for

the

enhanced

chemiluminescence of luminol/H2O2 system is quite sensitive to



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Page 16 of 41

the sizes of the Pt DENs in this range of sizes less than 3 nm, even with the sub-nanometer changes in size, as well as the generation

numbers

of

dendrimers

(i.e.

the

porosity

of

dendrimers) encapsulating the catalytic Pt nanoparticles. The good linearity of the enhanced chemiluminescence of luminol with the Pt DENs according to the concentration of H2O2 also suggests the feasible use of the Pt DENs for sensitive chemiluminescencebased applications to analyses involving H2O2. The feasibility of the Pt DENs for the sensitive analytical applications will be discussed later. The

size-dependent

catalysis

of

the

Pt

DENs

could

be

attributed to the difference in their surface areas since the heterogeneous catalytic reaction rate generally increases with the available surface area of catalysts.16 The larger surface area of the Pt DENs would be available with larger Pt DENs at the same given molar concentration of the Pt DENs, leading to better catalysis with the larger Pt DENs, i.e. G6-NH2(Pt200) DENs, in the present study as shown in Figures 2 and 3a. Therefore, we normalized the chemiluminescence to the surface area of the Pt DENs to compare the catalytic activities of the DENs having different sizes directly without considering the surface area effect.

Specifically,

we

determined

the

normalized

chemiluminescence by dividing the chemiluminescence emission by the surface area of the Pt DENs. The surface areas of the Pt


Page 17 of 41

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DENs were determined based on the particle sizes, which were experimentally measured by TEM, of the Pt DENs by assuming the spherical shape of the Pt nanoparticles. Figure 3b shows the normalized chemiluminescence obtained with the Pt DENs having three different sizes (i.e. G6-NH2(Pt55), G6-NH2(Pt147), and G6NH2(Pt200)

DENs),

normalized

according

to

the

chemiluminescence

concentration

of

H2O2

concentration

increases

increases

for

of

H2O2.

The

as

the

DENs.

More

linearly the

Pt

importantly, the Pt DENs of different sizes exhibited different slopes

in

the

increase

of

the

normalized

chemiluminescence,

which indicates the different catalytic activities of the Pt DENs even after eliminating the surface area effect. Especially, the

G6-NH2(Pt200)

DENs

displayed

the

steepest

increase

in

the

normalized chemiluminescence as the H2O2 concentration increased, which indicates the highest specific activity of the G6-NH2(Pt200) DENs

for

system

the

among

enhanced the

Pt

chemiluminescence

DENs.

These

of

results

the

luminol/H2O2

indicate

that

the

increased catalytic activities of the Pt DENs as the size of Pt nanoparticles increases could not be solely attributed to the increased surface area of the Pt nanoparticles. To

acquire

activities

of

further

the

Pt

insight

DENs

into

depending

the on

different the

size

catalytic of

the

Pt

nanoparticles, we employed XPS measurements of the Pt DENs and the

Pt2+

ion-dendrimer

complexes.

Figure


4

shows

the

Pt(4f)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 41

region in the XPS spectra of the Pt2+ ion-dendrimer complex with the

Pt2+/G6-NH2

ratio

of

200

G6-NH2(Pt2+)200),

(i.e.

as

a

representative of the Pt2+ ion-dendrimer complexes, and the Pt DENs

having

three

different

sizes

(i.e.

G6-NH2(Pt55),

G6-

NH2(Pt147), and G6-NH2(Pt200)). As shown in Figure 4a, the Pt(4f7/2) and Pt(4f5/2) peaks for the G6-NH2(Pt2+)200 complex appear at 72.0 eV and 75.3 eV, respectively. These values correlate to those reported for the PtCl42- starting material (i.e. 73.2 eV Pt(4f7/2) and 76.5 eV Pt(4f5/2)).33 The slight shift to a lower binding energy

for

the

G6-NH2(Pt2+)200

complex

suggests

the

strong

complexation of the Pt2+ ions to the dendrimer binding sites (i.e. the interior amines of the dendrimer) in the complex.34 In contrast, the Pt(4f) XPS spectra of the Pt DENs, which were obtained by reduction of the Pt2+ ion-dendrimer complexes with the addition of NaBH4, exhibit two pairs of peaks, indicating two populations of Pt oxidation sates in the Pt DENs. The new pair of Pt(4f7/2) and Pt(4f5/2) peaks at lower binding energy (shown as the vertical solid lines in Figure 4b) correspond to the reduced zerovalent

Pt

dendrimers.21,35

nanoparticles

Note

that

the

encapsulated

zerovalent

Pt

inside

nanoparticles

the are

responsible for the catalytic activity of the Pt DENs for the enhanced

chemiluminescence

of

the

luminol/H2O2

system

as

discussed earlier. In addition to these zerovalent Pt(4f) peaks, the other set of Pt(4f) peaks, corresponding to the unreduced


Page 19 of 41

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Pt2+ ion-dendrimer complex, are also present, as shown by the vertical Pt(4f)

dotted

peaks

lines

of

in

Figure

4,

G6-NH2(Pt2+)200

the

correlating

complex

well

observed

to

prior

the to

reduction. These results indicate the partial reduction of the Pt2+ ion-dendrimer complexes by NaBH4 during the synthesis of the Pt DENs, which is consistent with the previous finding by Crooks et

al.

that

the

NaBH4

reduction

leads

to

only

the

partial

reduction of the precursor complexes for the synthesis of Pt nanoparticles

encapsulated

inside

hydroxyl-terminated

dendrimers.34 Note also that the catalytic activity of the Pt2+ ion-dendrimer complexes is negligible compared to that of the Pt DENs, as discussed earlier. Interestingly, we found that the Pt DENs having three different sizes exhibit different extents of reduction for each Pt DEN. The deconvoluted Pt(4f) XPS spectra of the Pt DENs indicate that the extent of reduction is 29%, 36%, and 61% for the G6-NH2(Pt55), G6-NH2(Pt147), and G6-NH2(Pt200) DENs,

respectively

quantitative

(Supporting

extents

of

Information,

reduction

were

Figure

S5).

The

determined

from

the

deconvolution of the XPS spectra into peaks corresponding to the zerovalent Pt nanoparticles and the unreduced Pt2+ ion-dendrimer complexes.

Therefore,

these

observations

indicate

that

the

higher specific activity of the Pt DENs as the size of the Pt nanoparticles

increases

reduction

the

in

Pt

results DENs

with

from

the

larger


increased sizes

and

extent

of

thus

the


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

corresponding

increase

in

the

Page 20 of 41

relative

amount

of

catalytic

zerovalent Pt nanoparticles encapsulated inside the dendrimers. Importantly, this finding suggests that the dependence of the catalytic properties of the Pt DENs on their particle sizes originates from the different oxidation states of Pt as well as from structural effects such as surface area, which might be more dramatic for the very small nanoparticles in the size range of less than 3 nm. As verified thus far, the Pt DENs exhibited significant catalytic

activity

for

the

enhanced

chemiluminescence

of

the

luminol/H2O2 system. It is widely accepted that the generation of chemiluminescence

of

luminol

the

to

yield

aminophthalate

luminol/H2O2 emitting

anions)

via

involves

species

the

(i.e.

oxidation

the

pathways.36

multistep

excited The

of 3-

luminol

oxidation process is also believed to be facilitated by reactive oxygen species, such as OH·, O2·–, and

1

O2, generated from the

decomposition of H2O2.16,29 Thus, we hypothesized that the enhanced chemiluminescence of luminol/H2O2 could arise from the catalyzed generation of the reactive oxygen species (i.e. OH·, O2·–, and 1

O2) in the presence of the Pt DENs. The catalyzed generation of

the

reactive

oxidation

oxygen

process

aminophthalate chemiluminescence.

species

for anions, This

the

would

facilitate

formation leading

hypothesis

of to

is


the the

plausible

the

luminol

excited

3-

enhanced since

the

Page 21 of 41

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chemiluminescence in the presence of the Pt DENs originates from the

same

anions)

emitting as

species

discussed

(i.e.

the

earlier.

To

excited prove

3-aminophthalate the

hypothesis

experimentally, we carried out a set of quenching experiments of the chemiluminescence of luminol/H2O2 in the presence of the Pt DENs using specific quenchers for the OH·, O2·–, and

1

O2. First,

we observed the dramatic change in the chemiluminescence in the presence of the G6-NH2(Pt200) DENs after the addition of thiourea, sodium

salicylate,

quenchers

for

and

OH·.

As

sodium

benzoate,

shown

in

which

Figure

are

S6

specific

(Supporting

Information), the addition of the sufficient amount of thiourea, sodium

salicylate,

and

sodium

benzoate

resulted

in

the

significant inhibition of the chemiluminescence of luminol/H2O2, even in the presence of the Pt DENs. Similarly, the addition of ascorbic

acid

and

specific

scavengers

nitrotetrazolium for

O 2 ·–

as

blue

well

chloride,

as

OH·,

which

are

quenched

the

original chemiluminescence emission substantially. These results suggest that the enhanced chemiluminescence obtained with the G6-NH2(Pt200) DENs is attributed to the presence of the OH· and O2 ·–

during

the

chemiluminescence

process.

Furthermore,

the

addition of sodium azide (NaN3), which is a specific quencher for 1

O2, also effectively inhibited the enhanced chemiluminescence of

luminol/H2O2, which suggests the

1

O2–induced chemiluminescence in

the presence of the G6-NH2(Pt200) DENs. This is comparable with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the

previous

reports

chemiluminescence

of

Page 22 of 41

demonstrating

luminol/H2O2

1

the

in

the

O2–involved

presence

of

nanocatalysts such as carbon nanodots and graphene oxide.28,29 We further

confirmed

the

catalyzed

generation

of

1

O2

during

the

enhanced chemiluminescence of luminol/H2O2 in the presence of the G6-NH2(Pt200) in the ESR studies using TEMP as a 1O2-specific probe (Supporting Information, Figure S7). It is also possible that oxygen dissolved in the aqueous reaction solutions was involved in

the

generation

formation

of

of

O2·–.16

the

enhanced

Indeed,

the

chemiluminescence

via

deaeration

aeration

and

the

conditions during the measurements of the chemiluminescence in the presence of the G6-NH2(Pt200) DENs led to a 17% decrease and 8% increase, respectively, in the original chemiluminescence. However,

it

is

worth

noting

that

the

changes

in

the

chemiluminescence intensity under the deaeration and aeration conditions majority

of

were

relatively

reactive

oxygen

small, species

which

suggests

that

the

are

generated

from

H2O2

rather than O2. Taken all together, the set of the quenching experiments,

the

ESR

studies,

and

the

deaeration/aeration

experiments strongly suggest that the catalyzed generation of the reactive oxygen species including OH·, O2·–, and

1

O2, which

are primarily from H2O2, is responsible for the highly enhanced chemiluminescence of luminol/H2O2 in the presence of the Pt DENs (Supporting Information, Figures S6 and S7).


Page 23 of 41

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Facile and versatile applications of Pt DENs for sensitive chemiluminescence-based

analyses.

Taking

advantage

of

the

significant catalytic activity of the Pt DENs for the enhanced chemiluminescence of luminol/H2O2, we demonstrated the feasible use of the Pt DENs for sensitive analytical applications. Since the

enhanced

chemiluminescence

of

luminol

exhibited

good

linearity in the intensity according to the concentration of H2O2, as briefly discussed earlier, the Pt DENs would be useful especially for sensitive chemiluminescence-based applications to analyses involving H2O2. To demonstrate the feasibility of the Pt DENs

(particularly

G6-NH2(Pt200)

because

its

activity

is

the

highest among the Pt DENs explored in the present study) in sensitive effects

analytical of

the

applications,

reaction

we

first

conditions

investigated

(including

the

the pH,

concentration of luminol, and concentration of G6-NH2(Pt200) DEN) on the enhanced chemiluminescence of the luminol/H2O2 system in the presence of the G6-NH2(Pt200) DENs (Supporting Information, Figure

S8).

Figure

S8a

shows

that

the

chemiluminescence

intensity of luminol/H2O2 is dependent on the pH values of the reaction

solution

containing

the

G6-NH2(Pt200)

DENs,

which

is

consistent with previously reported nanoparticles enhancing the chemiluminescence chemiluminescence

of

luminol/H2O2.16,17

intensity

reached

its

For maximal

example,

the

values

under

strong alkaline conditions, specifically at pH 13 or 11.5 with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

gold

or

cupric

oxide

Page 24 of 41

respectively.37,38

nanoparticles,

However,

compared to the previously reported nanoparticle catalysts, the maximum chemiluminescence was achieved under a relatively weak alkaline which

condition

would

make

(i.e. the

pH

9.5)

DENs

more

with

the

beneficial

G6-NH2(Pt200) for

their

DENs, direct

applications in the analysis of biological samples that require mild assay conditions.39 The chemiluminescence of luminol/H2O2 was also

dependent

Figure

S8b.

on

the

The

concentration

of

luminol

chemiluminescence

as

intensity

shown

in

increased

significantly with the increase of luminol concentration up to 5 mM, and then reached a steady level. In addition, Figure S8c shows

that

the

chemiluminescence

intensity

increased

as

the

concentration of G6-NH2(Pt200) DEN increased up to 1 µM, which is also in agreement with other Pt DENs (Supporting Information, Figure S9). In

addition

to

the

effects

of

the

chemiluminescence

reaction conditions, we also investigated the robustness in the catalytic

activity

of

the

G6-NH2(Pt200)

DENs

for

the

enhanced

chemiluminescence of luminol/H2O2. The G6-NH2(Pt200) DENs exhibited robust

catalytic

compared

to

horseradish Figure

activity

for

conventional

peroxidase

S10).

chemiluminescence

Figure

the

enhanced

biological

(HRP)

enzymes

S10

shows

obtained

after

heat

chemiluminescence

catalysts

(Supporting the


as

Information,

change

treatment

such

of

in

the

the

G6-

Page 25 of 41

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NH2(Pt200) DENs and the HRPs at 95 °C for a range of incubation times. The decrease in the intensity was relatively negligible (i.e. 8%) with the G6-NH2(Pt200) DENs even after the harsh heat treatment of the DENs for up to 60 min compared to the sudden decrease of 78% obtained after the treatment of the HRPs for only 10 min. The negligible decrease in the chemiluminescence with the G6-NH2(Pt200) DENs indicates the excellent sustainability of the catalytic activity of the DENs even after their exposure to the harsh heat treatment. The excellent thermal stability of the

G6-NH2(Pt200)

chemiluminescence

was

also

confirmed

intensity-time

in

profiles

the of

dynamic

luminol/H2O2

obtained with the heat-treated G6-NH2(Pt200) and HRP (Supporting Information,

Figure

S11).

These

results

clearly

indicate

the

desirable stability and reliability of the G6-NH2(Pt200) DENs as efficient

catalysts

for

the

enhanced

chemiluminescence

of

luminol/H2O2, which makes the Pt DENs suitable for a broad range of analytical applications to analyses involving H2O2. On the basis of the advantages of the Pt DENs for the sensitive chemiluminescence-based applications, we demonstrated the versatile use of the DENs in oxidase-based analyses for the detection

of

various

oxidase

substrates.

Since

a

variety

of

oxidases generate H2O2 as a product via the oxidase-involving reactions of enzymatic substrates,40 the Pt DENs can be used to detect

the

oxidase

substrates

by


monitoring

the


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chemiluminescence

of

luminol

Page 26 of 41

triggered

by

the

biocatalyzed

generation of H2O2. Specifically, this was exemplified in the present

study

for

the

analysis

of

choline,

glucose,

and

cholesterol. In typical experiments for the analysis of choline, choline oxidase catalyzes the oxidation of choline to betaine with

the

concomitant

generation

of

H2O2,

and

the

oxidase-

generated H2O2 triggers the chemiluminescence of luminol in the presence of the G6-NH2(Pt200) DEN (See the Experimental Section for details). Choline is the precursor of the neurotransmitter, acetylcholine, which triggers neural responses. Hydrolysis of acetylcholine to choline is involved in the pathway regulating the neural responses.40 As shown in Figure 5a, we obtained a calibration curve for the chemiluminescence-based analysis of choline

with

exhibits

a

the

G6-NH2(Pt200)

good

chemiluminescence

DENs.

linear

intensity

The

calibration

relationship and

the

curve

between

choline

the

concentration

ranging up to 200 µM with a correlation coefficient of 0.98, which allows the limit of detection (LOD) of choline to be as low as 1.86 µM.41 Similarly, the use of the G6-NH2(Pt200) DENs also enabled

us

sensitive substrates

to

perform

other

oxidase-based

chemiluminescence-based including

glucose

detection and

analyses of

cholesterol

the

for

the

oxidase

with

the

corresponding oxidases. Figures 5b and 5c show the calibration curves for the chemiluminescence-based detection of glucose and


Page 27 of 41

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cholesterol, respectively. The LOD of glucose and cholesterol was

also

determined

respectively.41

to

These

chemiluminescence-based are

be

comparable

as

as

7.11

analytical assays

to

low

and

features

utilizing

other

µM

the

conventional

2.93

µM,

of

the

G6-NH2(Pt200) methods

DENs

for

the

determination of choline, glucose, and cholesterol,42-44 but could be

improved

conditions reaction

further such

buffer,

by

optimizing

the

oxidase-based

as

enzyme

concentration,

and

so

Most

on.

assay

incubation

importantly,

these

time, results

clearly demonstrate the facile and versatile applications of the Pt

DENs

in

the

sensitive


analyses

as

catalysts.

Conclusion In conclusion, we reported the synthesis of uniform Pt DENs having well-defined sizes over the range of 1 ~ 3 nm with subnanometer accuracy and the highly enhanced chemiluminescence of the

luminol/H2O2

activity

of

the

system Pt

with DENs,

the which

size-dependent

catalytic

facilitates

versatile

applications of the Pt DENs in diverse oxidase-based assays. Since

the

oxidase-based

assays

could

be

coupled

with

other

enzyme assays as demonstrated previously for the analysis of acetylcholine esterase inhibitors,40 the broader application of the Pt DENs is reasonably envisioned by the coupling of the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 41

oxidase-based assays demonstrated in the present study with a variety of enzymes. Interestingly, this study also illustrates that

the

chemical

states

of

nanoparticles

is

a

critically

important factor in the size-dependent catalysis of nanoscale catalysts as well as surface areas of the nanoparticles, which could

provide

a

useful

insight

for

the

design

of

efficient

nanoparticle catalysts for sensitive analytical applications.

Acknowledgments This work was supported by the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (NRF-2014S1A2A2028540 and NRF-2014R1A1A2058218), and the Agency for Defense Development through Chemical and Biological Defense Research Center

ASSOCIATED CONTENT Supporting Information Additional data including particle size distribution, UV-visible spectra,

chemiluminescence

intensity-time relative

chemiluminescence

scavengers, depending

profiles,

on

ESR pH

spectra, and

spectra,

dynamic

deconvoluted of

chemiluminescence

Pt(4f)

XPS

luminol/H2O2

chemiluminescence

concentrations

of

spectra,

after of

luminol

adding

luminol/H2O2 and

Pt

DENs,

relative chemiluminescence of luminol/H2O2 after heat treatment of


Page 29 of 41

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G6-NH2(Pt200) and HRP: These materials are available free of charge

via the Internet at http://pubs.acs.org.



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Page 30 of 41

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Page 32 of 41

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Figure Captions Figure 1. TEM images of as-synthesized (a) G6-NH2(Pt55), (b) G6NH2(Pt147), (c) G6-NH2(Pt200), and (d) G4-NH2(Pt55) DENs.

Figure

2.

obtained

Chemiluminescence

with

(i)

spectra

G6-NH2(Pt200),

(ii)

of

luminol/H2O2

G6-NH2(Pt147),

system

(iii)

G6-

NH2(Pt55), (iv) G4-NH2(Pt55), (v) G6-NH2(Pt2+)200, (vi) no Pt DENs, and (vii) G6-NH2 dendrimer. Conditions: 5 mM luminol, 1 mM H2O2, 500

nM

Pt

DENs,

500

nM

G6-NH2(Pt2+)200,

and

500

nM

G6-NH2

dendrimer.

Figure

3.

(a)

Chemiluminescence

emission

of

luminol

obtained

with (i) G6-NH2(Pt200), (ii) G6-NH2(Pt147), (iii) G6-NH2(Pt55), (iv) G4-NH2(Pt55), and (v) no Pt DENs. (b) Normalized chemiluminescence of luminol obtained with (i) G6-NH2(Pt200), (ii) G6-NH2(Pt147), and (iii) G6-NH2(Pt55). Conditions: 5 mM luminol and 500 nM Pt DENs.

Figure 4. XPS spectra in the Pt(4f) region of (a) G6-NH2(Pt2+)200 and (b) G6-NH2(Pt200), G6-NH2(Pt147), and G6-NH2(Pt55).

Figure 5. Calibration curves obtained with G6-NH2(Pt200) DENs for the sensitive chemiluminescence-based analysis of (a) choline, (b) glucose, and (c) cholesterol. Insets show the expanded view of the low-concentration regions. Conditions: 5 mM luminol and


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500 nM G6-NH2(Pt200). The chemiluminescence intensity was measured at 440 nm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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for TOC only


Tailoring Catalytic Activity of Pt Nanoparticles Encapsulated Inside

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