Ultrahigh Sensitivity Multifunctional Nanoprobe for ... - ACS Publications

Subscriber access provided by BALL STATE UNIV

Article

Ultrahigh Sensitive Multifunctional Nanoprobe for the Detection of Hydroxyl Radical and Evaluation of Heavy Metal Induced Oxidative Stress in Live Hepatocyte Quanwei Guo, Yuxin Liu, Qi Jia, Ge Zhang, Huimin Fan, Lidong Liu, and Jing Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00306 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

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

Analytical Chemistry

Ultrahigh Sensitive Multifunctional Nanoprobe for the Detection of Hydroxyl Radical and Evaluation of Heavy Metal Induced Oxidative Stress in Live Hepatocyte

Quanwei Guo†, Yuxin Liu†, Qi Jia, Ge Zhang, Huimin Fan, Lidong Liu, Jing Zhou*

Department of Chemistry, Capital Normal University, Beijing, 100048, P. R. China E-mail: [email protected]; Tel: +86-010-68902491 † These author contribute equal to this work.

Abstract: Hydroxyl radical (⋅OH) is important markers of the progress of heavy metal induced oxidative stress. However, most of reported probes and detection methods cannot meet the need of monitoring the ⋅OH concentration within the whole progress because of the limited linear range. Besides, low detection limit, high sensitivity, and good selectivity was also required. In this study, an ultrahigh sensitive multifunctional nanoprobe (ICG-modified NaLuF4:Yb,Er) was developed to evaluate heavy metal induced oxidative stress by detecting ⋅OH concentration, with a colorimetric, upconversion luminescence, and photothermal stepped method. This method has a broad linear detection range, from 16 pM to 2 µM, and a low detection limit of 4 pM. Besides, the nanoprobe

ACS Paragon Plus Environment


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 2 of 29

showed less response to ions, amino acids, biomolecules, and other radical oxygen species (H2O2 and O2-) than ⋅OH. This highly selective, highly sensitive probe with a broad linear detection range has great potential utility for monitoring ⋅OH concentration in live hypatocyte within the progress of heavy metal induced oxidative stress, with probable in vivo applications in the future. Keywords: upconversion nanoparticles; ICG; hydroxyl radical; stepped detection method; oxidative stress

Introduction Heavy-metal poisoning has recently been a major threat to human for its high incidence, mortality, and fatality rate. By Fenton reaction, heavy metal can lead reactive oxygen species (ROS), overproducing, accumulating, and further inducing DNA-damaging when the redox balance of organisms is disturbed

1-4

. Among all

these ROS, hydroxyl radical (⋅OH) has the greatest oxidative capacity and hence the strongest of destruction. Therefore, the development of nanoprobes for ⋅OH detection with high sensitivity and selectivity is priority to monitor ⋅OH concentration within the progress of heavy metal induced oxidative stress 5,6. Rare-earth-based

upconversion

nanoparticles

(UCNP) have

recently been developed as a novel potential platform for


Page 3 of 29

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


luminescent biosensing, when excited with near-infrared (NIR) laser in low power intensity range

7-9

. They have the advantages of low

toxicity, excellent stability, high contrast in biosamples, and great depth of tissue penetration 10-14. Many research groups have reported many

UCNP-based

sensors

for

the

selective

detection

of

hypochlorous acid, glutathione, nucleic acid, cyanide (CN–), and methylmercury

15-19

cyanine-linked

dyes

.

Recent has

research

significant

have

shown

that

reaction

with

⋅OH,

changing/disturbing their characteristic absorption in the NIR region 20,21

. Indocyanine green (ICG) is a typical U.S. Food and Drug

Administration (FDA)-certified cyanine dye for safe application in vivo. 22,23 Because of the overlap between the NIR absorption of ICG (600–800 nm) and the red emission region of UCNP (∼654 nm), a colorimetric and fluorescence method based on ICG-modified UCNP could be expected to detect ⋅OH, and the combination of upconversion luminescence and colorimetry should detect ⋅OH in a broad range of concentrations, from nanomolar (nM) to micromolar (µM)

15-23

. However, ⋅OH concentration in live hypatocyte without

heavy metal poisoned was low and therefore a lower detection limit was required to monitor the ⋅OH concentration within the whole progress.



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 4 of 29

ICG has also been studied for many years as an extremely useful concentration-dependent photothermal conversion agent

24-26

. A

slight change in absorption directly influences the molar extinction coefficient and also affects the photothermal temperature change, suggesting that a photothermal-based method could achieve a lower detection limit compared than the colorimetric and upconversion luminescence method. Therefore, the detection of ⋅OH based on a photothermal method was also considered. In this study, a uniform ICG-modified NaLuF4:Yb,Er nanocomposite (UCNP-ICG) was designed as a probe with which to detect ⋅OH. Based on the specific reaction of ICG and ⋅OH, a novel stepped method with ultrahigh sensitivity and good selectivity is reported that combines colorimetry, upconversion luminescence, and photothermal detection method. Based on the discussed stepped method, the ICG-modified UCNP provide a novel probe for the sensitive and selective detection of ⋅OH, with a broad detection range and a low detection limit, allowing the whole progress of heavy-metal induced oxidative stress to be monitored and evaluated in live hepatocyte. (Scheme 1).

Experiment Section Synthesis of NaLuF4:Yb,Er nanoparticles (UCNP-ICG)


Page 5 of 29

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 UCNP was synthesized via a typical solvothermal method

27,28

.

After removing the OA ligand on UCNP by NOBF4, the UCNP was redissolved in deionized water for ICG modification

29

(Please see

Supporting Information for experiment detail). Novel stepped detection of ⋅OH 1 mg UCNP-ICG was dispersed in 1 mL phosphate buffer solution (PBS) and formed a homogeneous system with pH = 7.4. A series of ⋅OH solutions with concentrations from 1 pM to 10 µM were added into the homogeneous system, incubated for 10 minutes and protected from light. Then, the UV-vis-NIR and UCL spectra of all samples were determined, respectively and the temperature signal within two minutes laser irradiation (808 nm, 0.64 W cm-2) were collected. Detection of ⋅OH concentration in live hypatocyte Human normal hypatocyte (CCC-HEL-1) were seeded into a 96-well cell culture plate at 1 ×104 well-1 under 100% humidity, and were cultured at 37oC and 5% CO2 for 24 hours. The ⋅OH concentration of cells was detected by stepped method for over 4 hours. Then, different concentration of CdCl2 solution (resulting concentration: 20 µM or 20 nM) was added into wells, respectively. The ⋅OH concentration was monitored by stepped method every 30 minutes within 25 hours.



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 6 of 29

Results and Discussion Design and characterization Oleic acid (OA)-coated NaLuF4:Yb,Er nanoparticles (UCNP) were synthesized with the typical solvothermal method.

28,29

Transmission

electron microscopic (TEM) images indicated that the UCNP has good dispersion, high crystallinity, and uniform spherical shapes, with a narrow diameter distribution of ∼40 nm (Figure 1A,C). A powder X-ray diffraction (XRD) pattern analysis (Figure 1B) and selected-area electron diffraction (SAED) (Figure 1A, bottom inset) of UCNP showed a hexagonal phase, based on the diffraction angle and interplanar distance (JPCDS: 027-0726). The energy-dispersive X-ray analysis (EDXA) spectrum confirmed the exclusive particle composition of sodium (Na), lutetium (Lu), ytterbium (Yb), and fluorine (F) (Figure 1D). Because the dopant content of erbium (Er) was < 2%, the peaks of Er could not be seen in the EDXA spectrum. Ligand-free UCNP were generated with a ligand-exchange reaction, for further modification. The OA-coated UCNP were treated with nitrosonium tetrafluoroborate (NOBF4) to remove the hydrophobic ligands from the UCNP, and the particles were then dispersed in deionized water. ICG was then modified on the surfaces of the ligand-free UCNP and the ICG loading amount was calculated to be 23.2 mg g-1. TEM images of UCNP-ICG suggested that no noticeable shape and size change


Page 7 of 29

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


can be observed before and after their modification with ICG (Figure S1A). The Fourier transform infrared (FTIR) spectrum of UCNP-ICG showed the same bonds as free ICG at 3232 cm–1 (R2−C=CH−R) and 1637 cm–1 (C=C in phenyl) (Figure S1B), and the ultraviolet–visible–NIR (UV–vis–NIR) spectra confirmed that UCNP-ICG showed the absorption peak characteristic of free ICG at 785 nm (Figure S1C). According to these results, ICG was successfully modified on the surfaces of UCNP. The ICG-modified UCNP also showed good solubility and good stability in

various

biological

solutions

(Figure

S2-4).

Zeta

potential

measurements showed a clear decline from +12 mV to –33 mV after ICG modification (Figure S1D), indicating that the modification was attributable to a charge-based interaction between the negatively charged sulfonate groups in ICG and the positively charged rare-earth ions on the UCNP surfaces 29,30. Novel stepped detection method It has been reported that ROS can oxidize large conjugated cyanine dyes via one-electron oxidation progress and the following polymethine chain cleavage, reducing their absorption in NIR region (scheme 1)23. To prove the interaction between UCNP-ICG and hydroxyl radical (⋅OH), the

optical

properties

of

UCNP-ICG

were

first

investigated.

Upconversion luminescence (UCL) spectrum of UCNP suggested the prominent UCL emission bands centered at 521, 540, and 654 nm in 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

visible region, which were readily assigned to the Er3+ transitions from the 2H11/2, 4S3/2, and 4F9/2 levels to the 4I15/2 ground state, respectively. The modified ICG as a quencher can efficiently decrease UCL emission intensity at 654 nm, due to the absorption band of ICG overlaps with the emissions area from 600 to 800 nm, hence enabling the generation of Förster resonance energy transfer (FRET) (Figure S5). Considering the fact that the oxidation product, widely known as oxindoles, showed weak absorption in NIR region and relatively strong absorption at 400-600 nm, the UCL emission intensity at 654 nm (red) would recover as ⋅OH involved while 540 nm emission (green) would be weakened (Scheme 1). Therefore, UCNP-ICG can be used as a probe to detect ⋅OH by colorimetric and upconversion luminescent method. The colorimetric detection method was first investigated by determining the UV–vis–NIR spectra of a mixture of UCNP-ICG (1 mg mL–1) in phosphate-buffered saline (PBS; pH = 7.4) and various concentrations of ⋅OH (1 pM–2 µM). The absorption at 400 nm and 785 nm were used to calculate the ratio of absorption intensity at 400 nm (I400) to the intensity at 785 nm (I785) (I400/I785) (Figure S6). As shown in Figure 2A, the probe showed a good response to ⋅OH as the concentration of ⋅OH increased. From 250 nM to 2 µM, the relationship between I400/I785 and the concentration of ⋅OH showed a good linear correlation (I400/I785 =


Page 8 of 29

Page 9 of 29

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


4.144 + 0.359 [⋅OH]/µM, R2 = 0.9981) with a detection limit of ∼4 pM (signal-to-noise ratio ≥ 3) (Figure 2B). The upconversion luminescent detection method based on UCNP-ICG was then tested. As shown in Figure 3A, the probe showed a good response to ⋅OH as the ⋅OH concentration increased. From 125 pM to 250 nM, the relationship between the ratio of the UCL intensity at 654 nm to the intensity at 540 nm (I654/I540) and the concentration of ⋅OH showed a good linear correlation (I654/I540 = 1.105 + 0.003 [⋅OH]/nM, R2 = 0.9972), with a detection limit of ∼100 pM (signal-to-noise ratio ≥ 3) (Figure 3B). It is interesting to find that the linear range of upconversion luminescent method (125 pM to 250 nM) can precisely connect with the linear range of colorimetric method (125 nM to 2 µM), which achieved a stepped detection of ⋅OH. More significantly, the stepped detection can efficiently decrease the detection limit and broaden detection range, which may provide a new way to develop novel detection method with low detection limit and broad detection range. Nevertheless, considering the low amount of ⋅OH in live hypatocyte without heavy metal poisoned, further low detection limit was necessary to monitor and evaluate the heavy metal induced oxidative stress. After carefully analyzing the UV-vis-NIR spectra, a significant phenomenon can be observed (Figure 2B). With ⋅OH added, the absorption of ICG in NIR decrease rapidly at low concentration range (1



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 10 of 29

pM to 32 nM) and then slowly in high concentration range (> 32 nM). Since the photothermal effect was closely related to the absorption, the weakened absorption should lead to a reduced photothermal effect. The following equation explains this more concisely:

ε=

AVNPs ρ N A LC

(1)

Because the volume and weight of the actual particles are considered, a slight absorption change would lead to a marked change in the molar extinction coefficient, which would affect the final photothermal temperature change. Therefore, we inferred that the photothermal detection method would allow a lower detection limit. As shown in Figure 4A, the probe showed a good response to ⋅OH as the concentration increased. From 16 pM to 250 pM, the relationship between the temperature change and the concentration of ⋅OH showed a good linear correlation (temperature change = 14.272 – 0.029[⋅OH]/pM, R2 = 0.9951) with a detection limit of ∼4 pM (signal-to-noise ratio ≥ 3) (Figure 4B). The irradiation time (within 2 min) had no obvious effect on the linear correlation (Figure S7). Photothermal detection based on UCNP-ICG showed a lower detection limit (pM) for ⋅OH than the other detection methods and a linear range (16 pM to 250 pM) connected precisely with upconversion luminescent method, successfully structuring a novel stepped method when combining with the colorimetric and upconversion


Page 11 of 29

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


luminescent method. Therefore, the stepped method, combining colorimetric, upconversion luminescent, and photothermal method, has a broad linear range of 16 pM (1.6×10-11 M) to 2 µM (2.0×10-6 M), and low detection limit down to 4 pM (4.0×10-12 M). Comparing with other reported method, the stepped method has relatively broader linear range and lower detection limit, which has great advantages in monitoring the ⋅OH concentration in live hypatocyte within the progress of heavy metal induced oxidative stress (Table 1) 31-34. Selectivity is an important factor when investigating the sensing properties of this novel stepped method. To better simulate the reaction in the physiological environment, we investigated the relative intensities of the UV–vis–NIR (Figure 2C), UCL (Figure 3C), and photothermal signals (Figure 4C) from other ions, amino acids, and biomolecules, including Na+, K+, Mg2+, Ca2+, Mn2+, Fe3+, Ni2+, Cl−, CO32−, PO43−, valine, glycine, cysteine, glutamic acid, lysine, glucose, glutathione, dopamine, and ascorbic acid, in the resultant solution. Since the reaction between ICG and ⋅OH is unique, none of the ions, amino acids, and biomolecules had an obvious effect in any of the three detection methods. Besides, other ROS which may be also produced by heavy metal were also taken for comparison to illustrate the selectivity. All the studied ROS, including hydroperoxide (H2O2) and superoxide anion (O2-), showed much weaker response than ⋅OH. Besides, the ⋅OH has no obvious influence on 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

optical properties (absorption, luminescence, and photothermal property) of UCNP (Figure S8). The above facts can demonstrate that UCNP-ICG has good sensitivity and selectivity for ⋅OH detection. ⋅OH concentration monitoring and oxidative stress evaluation Heavy-metal poisoning has received increasing attention in recent years with the development of heavy industries. Cadmium (Cd), which was widely used in battery and wear-resistant metal production, is usually considered a typical heavy metal, and potentially a major cause of ⋅OH overproduction. Cd2+ can permanently increase cellular ⋅OH signals by displacing endogenous Fenton metals from biomolecules, thus increasing the levels of free redox-active metals 4,35. Therefore, Cd2+ was used as the concentrate agent of ⋅OH in our experiment, which did not affect the detection results (Figure S9). Moreover, because UCNP-ICG can be used in the stepped method, which includes the photothermal method (pM), the UCL method (nM), and the colorimetric method (nM-µM), to detect ⋅OH detection in a broad concentration range (pM–µM) with a low detection limit (pM), it should be useful for monitoring ⋅OH concentration in live hypatocyte within the progress of heavy metal induced oxidative stress (Scheme 1). The ⋅OH concentration in cells was monitored for > 25 h before and after the addition of Cd2+ (resulting concentration, 20 µM) with the novel stepped method. As shown in Figure 5A, the ⋅OH concentrations


Page 12 of 29

Page 13 of 29

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


remained low (< 0.1 nM) in normal liver cells within the first 12 h (0–12 h), but increased progressively to 250 nM after Cd2+ was added. The Cd2+-induced overproduction of ⋅OH was clearly seen in the subsequent 13 h (13–25 h). The concentration of ⋅OH increased rapidly and remained at a high level, suggesting the over accumulation of ⋅OH in the cells. Interestingly, the rate of ⋅OH accumulation reached a plateau at 15–16 h. To study this phenomenon, the oxidative stress progress was initiated with the addition of a smaller amount of Cd2+ (resulting concentration, 20 nM). The concentration of ⋅OH increased to 150 nM within 4 h after the addition Cd2+ (12–16 h) and then decreased back to a low level (< 30 nM) (Figure 5B). We attributed these results to the redox balancing mechanism of organisms and their cellular defense mechanisms. When ⋅OH accumulate but remain at a low level, the redox balancing mechanisms of the organism and its cellular defense mechanisms can efficiently reduce the ⋅OH concentrations to protect the cells. However, at higher ⋅OH concentrations, the redox-balancing mechanisms of the organism and its cellular defense mechanisms may fail, ultimately leading to irreversible damage by the over accumulation of ⋅OH. Based on above results, the ⋅OH concentration in unpoisoned live hypatocyte was low and increasing rapidly within the progress of heavy metal induced oxidative stress with a plateau, which contributed to the redox balancing mechanisms. Further study showed that there were



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

distinct change features after the plateau when different amount of heavy metal involved. Importantly, we discovered that the ⋅OH concentration before Cd2+ involved was down to ~110 pM and the plateau, in this study, was calculated to be 118 nM (20 nM Cd2+) to 187 nM (20 µM Cd2+), which confirmed to the linear range of photothermal method (16 pM to 250 pM) and upconversion luminescent method (125 pM to 250 nM), respectively. With high concentration Cd2+ (20 µM) involved, the ⋅OH concentration would continuously increase to > 300 nM after the plateau which fit the colorimetric linear range (125 nM to 2 µM). Therefore, we assumed that the stepped method can provide a grade evaluation to cells via a binary system (Table S1). When the ⋅OH concentration was within the linear range of the detection method, it was defined as “1”. Otherwise, it was defined as “0”. A concentration of “1” detected with the photothermal method suggests that the liver cells are normal and safe. A concentration of “1” detected with the UCL method still suggests that the cells are normal, but are in an intermediate state, because the ⋅OH concentration is obviously increasing and the redox-balance still works. A concentration of “1” detected with the colorimetric method or when all the detection methods show a concentration of “0” suggests the over accumulation of ⋅OH in the cells, which will result in irreversible damage.


Page 14 of 29

Page 15 of 29

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


To further investigate the performance of UCNP-ICG and the novel stepped method in monitoring the ⋅OH concentration in live hypatocyte, the recovery rate was calculated with the following equation:

P =（c2－c1）/ c3 × 100%

(2)

Where P is the recovery rate, c2 is the measured value, c1 is the concentration of ⋅OH in normal liver cells (average = 0.847 nM), and c3 is the concentration of ⋅OH in the added samples (Equation 2). With this equation, the recovery rate of the stepped method was calculated to be 99.29 ± 2.05% (α = 0.90), which suggests that the novel stepped method has a high recovery rate and can be used to monitor ROS concentrations in live hypatocyte within the progress of heavy metal induced oxidative stress (Figure S10). Toxicity studies Although ICG has been approved to be safe for clinical application in patient by the U.S. FDA for many years, we also checked the cytoxicity and in vivo toxicity of UCNP-ICG. A high cellular viabilities of ∼90% can

be

calculated

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

from bromide

(MTT)

assay, suggesting that there was no significant viability difference of HCT116 cells (cancer cells) and CCC-HEL-1 cells (normal cells) when they were incubated with/without the presence of UCNP-ICG (0–1.0 mg mL–1) within 24 and 48 h. The MTT assay results can demonstrate a low ACS Paragon Plus Environment


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

cytotoxicity of UCNP-ICG (Figure S11). Moreover, toxicity was further studied in vivo. Healthy mice were injected intravenously with 20 mg kg–1 UCNP-ICG. No mouse death or has obvious abnormal behavior in the mice group which receive high dosage of UCNP-ICG. Complete blood panel tests and serum biochemistry assays were performed for the UCNP-ICG-injected mice after 1 h (Figure S12A), 7 days (Figure S12B), and 30 days (Figure S12C). The liver function indices, including alanine aminotransferase, aspartate aminotransferase, total bilirubin, total protein, and albumin, and the kidney function indicator, creatinine, were all normal, suggesting that no obvious damage to liver or kidney was induced by UCNP-ICG. A hematoxylin–eosin stained histological examination illustrated that UCNP-ICG has no perceptible adverse effects on the examined major organs within 30 days, including the heart, liver, spleen, kidney, and lung (Figure S12D). Our results demonstrate that UCNP-ICG is not obviously toxic to mice after its systemic administration at the dose tested.

Conclusion In summary, by exploiting the unique reaction between ICG and ⋅OH, ICG-modified UCNP were designed as a probe with which to detect ⋅OH. By combining UCL, UV–vis–NIR, and photothermal temperature signals, a novel stepped detection method was developed. The stepped method


Page 16 of 29

Page 17 of 29

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


has a broad linear detection range of 16 pM to 2 µM, with a low detection limit of 4 pM. The ⋅OH concentration in live hepatocyte via heavy metal inducing was monitored with this novel stepped method. The probe showed no obvious toxicity to living systems. This study not only provides a new stepped method for the detection of ⋅OH across a broad linear range of concentrations, high selectivity and sensitivity, and a high recovery rate, but also provides a novel analytical platform for monitoring heavy-metal-induced ⋅OH accumulation in live hepatocyte.

Acknowledgements The authors thank Prof. Yuqing Lin from Capital Normal University for discussing the results. The authors also thank the funding of National Natural Science Foundation of China (21301121), Beijing talent foundation outstanding young individual project (2015000026833ZK02), the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality (IDHT20140512), Scientific Research Base Development Program of the Beijing Municipal Commission of Education (KM201410028008), and Beijing

Municipal

Science

&

Technology

Commission

(Z131103002813097).

Supporting information availiable: Detailed experiment section information.



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

TEM image, FTIR spectra, UV-vis-NIR spectra and zeta potential of UCNP-ICG; UCL spectrum of UCNP and the UV-vis-NIR absorption of ICG; Absorbance of ICG at 400 nm and 785 nm; Linear correlation between temperature change and ·OH concentration; Relative I(400 nm)/I(785 nm), relative I(654 nm)/I(540 nm), and relative temperature change of UCNP-ICG and UCNP; Photothermal, UCL, and UV-vis-NIR response of pretreated UCNP-ICG solution with 16 pM ·OH in the presence of Cd2+; The binary system based on stepped method for evaluation heavy metal induced oxidative stress in live hypatocyte; The recovery rate of the colorimetric, UCL, and photothermal detection method; MTT assay, serum biochemistry results and H&E-stained tissue sections of UCNP-ICG; ·OH concentration in hepatocyte.

Reference (1) Pelicano H.; Carney D.; Huang P. Drug Resist. Update 2004, 7, 97-110. (2) Fruehauf J.; Meyskens F. Clin. Cancer. Res. 2007, 13, 789-794. (3) Nogueira V.; Hay N. Clin. Cancer. Res. 2013, 19, 4309-4314. (4) Thevenod F.; Lee W. Arch. Toxicol. 2013, 87, 1743-1786. (5) Beibei L.; Peter L.; Neil V. Anal. Chem., 1997, 69, 4295–4302. (6) Li Q.; Velde C.; Israelson A; Xie J.; Bailey A.; Dong M.; Chun S.; Roy T.; Winer L.; Yates J.; Capaldi R.; Cleveland D.; Miller T. Proc. Natl. Acad. Sci. USA 2010, 107, 21146-21151.


Page 18 of 29

Page 19 of 29

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


(7) Zhou J.; Liu Q.; Feng W.; Sun Y.; Li F. Chem. Rev. 2015, 115, 395-465. (8) Zheng X.; Zhu X.; Lu Y.; Zhao J.; Feng W.; Jia G.; Wang F.; Li F.; Jin D. Anal. Chem. 2016, 88, 3449–3454. (9) Muhr V.; Wilhelm S.; Hirsch T.; Wolfbeis O. Acc. Chem. Res. 2014, 47, 3481-3493. (10) Liu X.; Yan C.; Capobianco J. Chem. Soc. Rev. 2015, 44, 1299-1301. (11) Wang X.; Chen J.; Zhu X.; Chen X.; Yan X. Anal. Chem. 2013, 85, 10225–10231 (12) Wu Y.; Zhang X.; Zhang D.; Zhang C.; Li J.; Wu Y.; Song Z.; Yu R.; Tan W. Anal. Chem. 2016, 88, 1639–1646 (13) Chen G.; Qiu; H.; Prasad P.; Chen X. Chem. Rev. 2014, 114, 5161–5214. (14) Liu Y.; Tu D.; Zhu H.; Chen X. Chem. Soc. Rev. 2013, 42, 6924-6958. (15) Hlaváček A.; Farka Z.; Hübner M. Horňáková V.; Němeček D.; Niessner R.; Skládal P.; Knoppand D.; Gorris H. Anal. Chem. 2016, 88, 6011–6017. (16) Deng R.; Xie X.; Vendrell M; Chang Y.; Liu X. J. Am. Chem. Soc. 2011, 133, 20168-20171. (17) Pakkila H.; Yliharsila M.; Lahtinen S.; Hattara L.; Salminen N.;



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

Arppe R.; Lastusaari M.; Saviranta P.; Soukka T. Anal. Chem. 2012, 84, 8628−8634. (18) Zhang F.; Haushalter R.; Haushalter R.; Shi Y.; Zhang Y.; Ding K.; Zhao D.; Stucky G. Small 2011, 7, 1972-1976. (19) Liu Y.; Chen M.; Cao T.; Sun Y.; Li C.; Liu Q.; Yang T.; Yao L.; Feng W.; Li F. J. Am. Chem. Soc. 2013, 135, 9869-9876. (20) Oushiki D.; Kojima H.; Terai T.; Arita M.; Hanaoka K.; Urano Y.; Nagano T. J. Am. Chem. Soc. 2010, 132, 2795-2801. (21) Zou X.; Liu Y; Zhu X.; Chen M.; Yao L.; Feng W.; Li F. Nanoscale 2015, 7, 4105-4113. (22) Sheng Z.; Hu D.; Zheng M.; Zhao P.; Liu H.; Gao D.; Gong P.; Gao G.; Zhang P.; Ma Y.; Cai L. ACS Nano, 2014, 8, 12310-12322. (23) Liu Y.; Li L.; Guo Q.; Wang L.; Liu, D.; Wei, Z.; Zhou J. Theranostics 2016, 6, 1491-1505. (24) Cheng Q.; Liang C.; Wang C.; Liu Z. Adv. Mater. 2014, 27, 903-910. (25) Yang H.; Mao H.; Wan Z.; Zhu A.; Guo M.; Li Y.; Li X.; Wan J.; Yang X.; Shuai X.; Chen H. Biomaterials 2013, 34, 9124-9133. (26) Ma Y.; Tong S.; Bao G.; Gao C.; Dai Z. Biomaterials 2013, 34, 7706-7714. (27) Jalil R.; Zhang Y. Biomaterials 2008, 29, 4122-4128. (28) Liu Y.; Guo Q.; Zhu X.; Feng W.; Wang L.; Ma L.; Zhang G.; Zhou J.; Li F. Adv. Funct. Mater. 2016, 26, 5120-5130.


Page 20 of 29

Page 21 of 29

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


(29) Ashokan A.; Gowd G.; Somasundaram V.; Bhupathi A.; Peethambaran R.; Unni A.K.K.; Palaniswamy S.; Nair S.V.; Koyakutty M. Biomaterials 2013, 34, 7143-7157. (30) Sharma P.; Bengtsson N.; Walter G.; Sohn H.; Zhou G.; Iwakuma N.; Zeng H.; Grobmyer S.; Scott E.; Moudgil B. Small 2012, 8, 2856-2868. (31) Liu F.; Du J.; Song D.; Xu M.; Sun G. Chem. Comm. 2016, 52, 4636-4639. (32) Huang W.; Xie W.; Shi Y.; Luo H.; Li N. J. Mater. Chem. 2012, 22, 1477-1481. (33) Yang Y.; Zhou J.; Zhang Y.; Zou Q.; Zhang X.; Chen J. Sensors Actuat. B-Chem. 2013, 182, 504-509. (34) Si J.; Lu L.; Gao Z.; Zhang Y.; Luo H.; Li N. Anal. Methods 2014, 6, 6536-6540. (35) Valko M.; Rhodes C.; Moncol J.; Izakovic M.; Mazur M. Chem-Biol. Interact. 2006, 160, 1-40.



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

Scheme 1. Working principle of multifunctional nanoprobe for the detection of ·OH and monitoring ·OH concentration in live hepatocyte within the progress of heavy metal induced oxidative stress.


Page 22 of 29

Page 23 of 29

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. The TEM image A). Insert: the scheme (top) and SAED pattern (bottom). XRD pattern B), Size distribution C), and EDXA D) of UCNP.



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 2. A) UV-vis-NIR spectra of standardized UCNP-ICG solution upon addition of various concentrations of ⋅OH. B) The linear relationship between I (400 nm) / I (785 nm) change with the ⋅OH concentrations in the range from 125 nM to 2 µM. C) UV-vis-NIR response of pretreated UCNP-ICG solution with 125 nM ⋅OH in the aqueous containing different final concentration of ions, amino acid, and biomolecules: 1 mM for Na+, K+, Mg2+, Ca2+, Mn2+, Fe3+, Cl-, CO32-, and PO43-. 1 µM for Val, Gly, Cys, Glu, Lys, Glc, GSH, DA, and AA. 125 nM for H2O2, and O2-.


Page 24 of 29

Page 25 of 29

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 3. A) UCL emission of standardized UCNP-ICG solution upon addition of various concentrations of ⋅OH. B) The linear relationship between I(654 nm) / I(540 nm) with the ⋅OH concentrations in the range from 125 pM to 250 nM. C) UCL response of pretreated UCNP-ICG solution with 125 pM ⋅OH in the aqueous containing different final concentration of ions, amino acid, and biomolecules: 1 mM for Na+, K+, Mg2+, Ca2+, Mn2+, Fe3+, Cl-, CO32-, and PO43-. 1 µM for Val, Gly, Cys, Glu, Lys, Glc, GSH, DA, and AA. 125 pM for H2O2, and O2-.



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 4. A) Photothermal temperature change of standardized UCNP-ICG solution upon addition of various concentrations of ⋅OH. B) The linear relationship between temperature change with the ⋅OH concentrations in the range from 16 pM to 250 pM. C) Photothermal response of pretreated UCNP-ICG solution with 16 pM ⋅OH in the aqueous containing different final concentration of ions, amino acid, and biomolecules: 1 mM for Na+, K+, Mg2+, Ca2+, Mn2+, Fe3+, Cl-, CO32-, and PO43-. 1 µM for Val, Gly, Cys, Glu, Lys, Glc, GSH, DA, and AA. 16 pM for H2O2, and O2-.


Page 26 of 29

Page 27 of 29

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 5. A) The ·OH concentration in live hypatocyte detected by novel stepped method within 25 hours with 20 µM A) and 20 nM B) Cd2+ involved. The first 12 hour in each experiment was Cd2+ free.



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

Table 1. Comparison with other reported method for detection of ·OH.


Page 28 of 29

Page 29 of 29

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


(For TOC only)


Ultrahigh Sensitivity Multifunctional Nanoprobe for ... - ACS Publications

Recommend Documents