Silver Doped Mesoporous Silica Nanoparticles Based Electrochemical Enzyme-Less Sensor for Determination of H₂O₂ Released from Live Cells

Abstract

In this study, a silver doped mesoporous silica nanoparticles-based enzyme-less electrochemical sensor for the determination of hydrogen peroxide (H₂O₂) released from live cells was constructed for the first time. The presented electrochemical sensor exhibited fast response (2 s) towards the reduction of H₂O₂ concentration variation at an optimized potential of −0.5 V with high selectivity over biological interferents such as uric acid, ascorbic acid, and glucose. In addition, a wide linear range (4 μM to 10 mM) with a low detection limit (LOD) of 3 μM was obtained. Furthermore, the Ag-mSiO₂ nanoparticles/glass carbon electrode (Ag-mSiO₂ NPs/GCE) based enzyme-less sensor showed good electrocatalytic performance, as well as good reproducibility, and long-term stability, which provided a successful way to in situ determine H₂O₂ released from live cells. It may also be promising to monitor the effect of reactive oxygen species (ROS) production in bacteria against oxidants and antibiotics.

1. Introduction

The rapid, specific, and accurate hydrogen peroxide (H₂O₂) determination is quite essential in bioanalytical fields. H₂O₂, as the most common representative of reactive oxygen species, which endogenously produced in a cell, is a key player in various pathological processes. High levels of H₂O₂ results in not only tissue and DNA damage, but also aging, diabetes, cancer, traumatic brain injury, and neurodegenerative disorders [1,2]. Consequently, the determination of H₂O₂ attracted much interest as its potential as an indicator of oxidative stress-related diseases. Several analytical methods such as spectrophotometry [3], fluorimetry [4], titrimetry [5], chromatography [6], and electrochemistry [7,8] have been applied for highly sensitive H₂O₂ determination. Amongst the above techniques, electrochemical methods are always optimal choices, due to their high sensitivity, operational simplicity, fast response, low cost, portability, and multi-analyte detection [9,10]. However, most electrode modifiers possessed high potentials, which limited the sensitive and selective performance of H₂O₂ at ordinary solid electrodes [11]. Thus, the electrode modification is of great importance to minimize the overpotential of redox reaction and improve the rate of electron transfer.

Reportedly, noble metal nanoparticles [12], noble metal nanoparticles on carbon nanostructures [13], and bimetallic nanoparticles [14] have been used for the electrode surfaces modification because of their unique catalytic and electronic properties. The constructed electrochemical sensors performed a highly electrocatalytic activity toward H₂O₂ reduction or oxidization. Compared with the enzyme-based sensor, enzyme-less sensors are specific to analyte and overcome the limitations from enzyme, such as high cost, susceptible conformation affected by pH, temperature, etc. [15]. Silver nanoparticles (Ag NPs) exhibit good synergetic effect on H₂O₂ reduction [16,17], which is widely used for enzyme-less electrochemical sensors of H₂O₂. However, Ag NPs are easy to aggregate in solution before making of electrodes, reducing the sensitivity of the electrochemical sensor. Use of mesoporous silica nanoparticles (mSiO₂ NPs) is one of the most effective ways to prevent aggregation [18] because Ag NPs can embed uniformly in the pores of mSiO₂ NPs. In addition, the mSiO₂ NPs possess unique properties such as large surface area, uniform pore size, ordered mesostructure, and facile surface modification [19,20], which make it a suitable electrically conductive host of Ag NPs to construct electrochemical sensors [21,22]. For example, Khan and Bandyopadhyaya [23] presented an enzyme-less amperometric H₂O₂ sensor based on Ag NPs impregnated amine functionalized mSiO₂ NPs with a wider linearity range (5.3–124.3 mM). Azizi et al. [24] reported a new Ag-doped SBA-16 NPs modified carbon paste electrode, which could achieve detection of H₂O₂ in two linear ranges (20 μM to 8 mM, and 8 to 20 mM). The biosensor constructed by Ensafi et al. [10] could achieve a lower LOD of 0.45 μM. While the significant success has been made in the detection of traces of H₂O₂, which is summarized in the new work by Viter and Iatsunskyi [25], biosensors based on Ag-doped MCM-41 mSiO₂ NPs for real-time detection of H₂O₂ released from live cells has not been reported yet, to the best of our knowledge.

We presented an enzyme-less electrochemical biosensor based on Ag-doped MCM-41 mSiO₂ NPs to determine H₂O₂ released from live cells (Figure 1). Our result showed a high electrocatalytic performance of Ag-mSiO₂ nanoparticles/glass carbon electrode (NPs/GCE) towards H₂O₂ reduction. Additionally, a good linear range from 4 μM to 10 mM with a LOD of 3 μM was obtained. The presented sensor showed high selectivity over biological interferents, such as ascorbic acid, uric acid, and glucose. Moreover, H₂O₂ released from pheochromocytoma (PC-12) cells could be successfully detected using the present sensor. In addition, as reactive oxygen species (ROS) including H₂O₂ production in bacteria such as Escherichia coli and Vaginal Lactobacilli can increase the bacteria’s susceptibility to oxidative attack from antibiotic treatment [26,27], our sensor is promising to help understanding the mechanism of H₂O₂ producing by bacteria against antibiotics in a clinic.

2. Materials and Methods

2.1. Materials

Tetraethylorthosilicate (TEOS), hexadecyltrimethylammonium bromide (CTAB), formalin (HCHO, 37%), and silver nitrate (AgNO₃) were purchased from Sigma Aldrich. N-(aminoethyl)-amino-propyl trimethoxysilane (TSD) was bought from Aladdin Company. Methanol (CH₃OH), disodium hydrogen phosphate (Na₂HPO₄·12H₂O), sodium dihydrogen phosphate (NaH₂PO₄·2H₂O), sodium hydroxide (NaOH), potassium chloride (KCl), potassium ferricyanide (K₃Fe(CN)₆), ammonium nitrate (NH₄NO₃), and ethanol (C₂H₅OH) were purchased from Beijing Chemical Plant. All chemicals were analytical grade. Distilled water (18.2 MΩ cm⁻¹) was used as a solvent.

2.2. Preparation of Ag-mSiO₂ Nanoparticles (NPs)

Ag-mSiO₂ NPs was prepared following slight modification of the Tian reaction [28]. A quantity of 0.5 mL 2M NaOH was added in 55 mL 7.5 mM CTAB aqueous solution to make solution A; 1 mL TEOS was added to 5 mL methanol to make solution B; 0.25 mL of TSD was added to 5 mL 35 mM AgNO₃ solution to make solution C. Under vigorous stirring, solution A was heated to 75 °C and added with 4 mL of solution B dropwise to react for 15 min. Then, solution C and 2 mL of solution B were added dropwise and reacted for 2 h. A total of 2.5 mL formalin solution was finally added and reacted for 1 h. The resulted NPs were removed from CTAB in NH₄NO₃ ethanol solution (50 mL) at 60 °C for 10 h under stirring. The resulted Ag-mSiO₂ NPs were centrifuged, washed with distilled water thrice, and re-suspended into 50 mL deionized water for further use.

2.3. Preparation of H₂O₂ Biosensors

The surface of a glassy carbon electrode (GCE, 3 mm in diameter) was firstly polished and cleaned to a mirror-like status. Different volumes of Ag-mSiO₂ NPs suspension (10, 8, 5, and 3 μL) were dropped respectively on the pretreated electrode surface and dried in the air for 1 h. Then GCE modified with a stable Ag-mSiO₂ NPs film (Ag-mSiO₂ NPs/GCE) was used for the following electrochemical experiments.

2.4. Determination of H₂O₂

2.4.1. Detection of Standard H₂O₂ Solution

H₂O₂ standard solutions with different concentrations were added to 10 mL of 0.2 M phosphate buffered saline (PBS, pH 6.8, saturated with N₂) under stirring, for H₂O₂ detection. The applied potential (−0.40, −0.45, −0.50, and −0.55 V) was optimized for amperometric analysis of H₂O₂. The background current was firstly recorded in PBS (pH 6.8) without H₂O₂ and subtracted for H₂O₂ calibration plot construction.

2.4.2. Detection of H₂O₂ Released from Pheochromocytoma cells

The pheochromocytoma (PC-12) cells were grown in 75 cm² flasks containing RPMI-1640 medium including fetal bovine serum (10%), as well as penicillin and streptomycin (100 μg·mL⁻¹) in 5% CO₂ atmosphere at 37 °C. When the PC-12 cells reached a 90% confluence growth, they were centrifuged and responded into 4 mL of PBS (0.2 M pH 6.8). The cell-packed pellet of 1.0–2.0 × 10⁵ cells·cm⁻² was obtained for amperometric analysis. After achieving a steady background noise, 60 µg·mL⁻¹ lipopolysaccharide (LPS) was added into the cell mixture to stimulate the release of H₂O₂ from cells. Then 500 unit·mL⁻¹ catalase was added to decomposing H₂O₂. As a control group, LPS and catalase were added to PBS solution without cells. An optimized potential of −0.5 V was applied to the Ag-mSiO₂ NPs-modified GC electrode for amperometric analysis.

2.5. Characterization

High-resolution transmission electron microscopy (HRTEM, CM200UT, Philips, FEI Co., Hillsboro, OR, USA) was used to examine the morphology and distribution of mSiO₂ NPs and Ag-mSiO₂ NPs. X-ray diffraction (XRD) pattern was analyzed by an X-ray powder diffractometer (D8 Advance, Bruker, Berlin, Germany). Ultraviolet-visible spectroscopy (UV-vis) absorbance spectra were recorded by UV-vis spectrophotometer (Nanjing Feile Instrument Company, Nanjing, China) with a wavelength range from 300 to 800 nm.

Cyclic voltammetric and amperometric analysis were performed on a CHI660E electrochemical analyzer (Chenhua Co., Shanghai, China) at room temperature. A bare Glassy Carbon (GC) electrode or Ag-mSiO₂ NPs modified GC electrode, a Platinum foil electrode and saturated calomel electrode (SCE), were used as the working, the counter and the reference electrode, respectively. Before the detection of H₂O₂, the solutions were purged by high purity N₂ for 30 min to remove oxygen.

3. Results

3.1. Characterization of Ag-mSiO₂ NPs

Figure 2A shows the UV-vis absorption spectra of mSiO₂ NPs and Ag-mSiO₂ NPs. The UV-vis absorbance spectrum of mSiO₂ NPs at 414 nm was enhanced after Ag NPs decorated. In the wide-angle XRD pattern of the Ag-mSiO₂ NPs (Figure 2B), four well-resolved diffraction peaks at 2θ values in the range of 30°–90° can be indexed to face-centered cubic (fcc) Ag 111, 200, 220, and 311 reflections [29]. The typical morphology and structure of mSiO₂ and Ag-mSiO₂ NPs was observed clearly through HRTEM. Small Ag NPs were doped in the ordered porous framework of spherical Ag-mSiO₂ NPs (Figure 2C,D), which protect themselves from aggregation.

3.2. Electrocatalytic Reduction of H₂O₂

The electrocatalytic activity of the Ag-mSiO₂ NPs modified GCE toward the H₂O₂ reduction was evaluated in PBS (0.2 M, pH 6.8) with a scan rate of 50 mV·s^-1. Different volumes of Ag-mSiO₂ NPs (3 μL, 5 μL, 8 μL, and 10 μL) modified on the electrode for better cyclic voltammetric (CV) responses were investigated. The modifier of 8 μL on the surface showed the best response, which was applied for the following experiment (Figure S1). The mechanism of H₂O₂ reduced by Ag-doped mesoporous NPs is as follows [24]:

• H₂O₂ + e⁻ ↔ OH_ads + OH⁻
• OH_ads + OH⁻ ↔ OH⁻
• 2OH⁻ + 2H⁺ ↔ 2H₂O

Figure 2A shows the cyclic voltammogram of bare GC electrode (curve a and c) and Ag-mSiO₂ modified GC electrode (curve b and d) in PBS (0.2 M, pH 6.8) with the absence or presence of 0.1 mM H₂O₂. In the absence of H₂O₂, no obvious cathodic current was observed for bare GCE or Ag-mSiO₂ modified GCE (Figure 3Aa,b). However, after the addition of H₂O₂, the reduction current of Ag-mSiO₂ modified GC electrode (Figure 3Ad) greatly increased, while the response signal of the bare GC electrode was very weak (Figure 3Ac). Cyclic voltammogram of Ag-mSiO₂ NPs modified GC electrode for H₂O₂ reduction at various concentrations of H₂O₂ was shown in Figure 3B. The result revealed that the current responses increased with an increased H₂O₂ concentration, indicating a good electrocatalytic behavior of Ag-mSiO₂/GCE towards H₂O₂ reduction.

3.3. Amperometric Determination of H₂O₂

The amperometric analysis was used to evaluate the electrocatalytic activity of Ag-mSiO₂/GC electrode for different concentrations of H₂O₂. First of all, the effect of applied potential on amperometric current-time (i−t) curve was studied. Several applied potentials of −0.40, −0.45, −0.50, and −0.55 V was employed to investigate the according amperometric response of Ag-mSiO₂/GC electrode in PBS (0.2 M, pH 6.8) with the successive addition of 1.0 mM H₂O₂. The amperometric response reached a maximum value at a potential of −0.50 V (Figure S2), which was selected for the following amperometric analysis. Figure 4A showed the amperometric i–t curves of Ag-mSiO₂ NPs/GC electrode with the successive additions of H₂O₂ into deoxygenated PBS (0.2 M, pH 6.8) under continuous stirring. Before adding H₂O_2, the amperometric current of Ag-mSiO₂ NPs/GC electrode was recorded in PBS solution for 200 s to obtain a steady blank control. The Ag-mSiO₂ NPs/GCE-based sensor could reach a steady-state current within 2 s after a small amount addition of H₂O₂, which indicated its fast response for H₂O₂ reduction. Additionally, a wide linear range from 4 μM to 10 mM was obtained. The LOD is calculated to be 3 μM (S/N = 3) (Figure 4B). The present H₂O₂ biosensor revealed a wide linear range, low LOD, and fast response.

3.4. Interferences Study

Glucose, ascorbic acid (AA), and uric acid (UA) are three commonly interferents in a physiological system. The anti-interference effect of the Ag-mSiO₂ NPs /GCE based sensor toward glucose, AA, and UA was investigated. The amperometric responses of the present sensor with sequential addition of 0.05 mM H₂O₂, 1 mM glucose, 0.15 mM AA, and 0.5 mM UA are shown in Figure 5. It can be seen that a large amperometric response was achieved with a low concentration of 0.05 mM H₂O₂, while negligible current was observed with high concentrations of the other interfering species such as glucose, AA or UA. This showed the present sensor was selective to H₂O₂.

3.5. Reproducibility and Stability

The reproducibility of Ag-mSiO₂ NPs modified single electrode was tested for five successive measurements with the relative standard derivation (RSD) in about 2.6%. Moreover, the RSD of current responses of five electrodes was investigated and found to be less than 5%, suggesting the high reproducibility and good precision of our present sensor. Furthermore, the current responses of the sensor decreased to 90.2% of its original value after being stored in the refrigerator at 4 °C for 20 days. The above results indicated the reliability and long-term stability of our fabricated H₂O₂ sensor. In addition, we compared the hydrogen peroxided sensing by AgNP impregnated silica NPs in

Table 1. Comparison of the present work with that reported in literature for hydrogen peroxide sensing by Ag NP impregnated silica NPs.

Types of Electrode	Applied Potential (V)	Linear Range (µM)	Detection Limit (µM)	Samples	Reference
AgNP-NH2-SBA-15-GCE	−0.4 vs. SCE	0.49–5.3 5.3–124.5	N.A.	PBS buffer	[19]
Ag/SBA-16/CPE	−0.45 vs. Ag|AgCl	20–8000 8000–20,000	2.95	Hair dying cream	[20]
Ag@SiO2 YSNs	−0.5 vs. Ag|AgCl	100–15,000	3.5	PBS buffer	[18]
Ag NPs/porous silicon	−0.45 vs. Ag|AgCl	1.65–500	0.45	Hair color oxidant	[10]
Nanoporous silver/Ni wire	−0.35 vs. SCE	10.0–8000	4.80	PBS buffer	[30]
Ag-mSiO2 NPs/GCE	−0.5 vs. SCE	4–10,000	3	Live cell	This work

. H₂O₂ sensor reported here has a wide linear range and low detection limit and can detect the H₂O₂ released from living cells.

3.6. Determination of H₂O₂ Released from Pheochromocytoma Cells

Moreover, we performed a study of the real-time determination of H₂O₂ released from live cells. For the experiment, about 2 × 10⁵ pheochromocytoma (PC-12) cells in 4 mL PBS (pH = 6.8) with 100 mM glucose were used. LPS (60 μg mL⁻¹) was employed to stimulate PC-12 cells to generate H₂O₂. After treating with LPS, an obvious current change occurred in solution with cells, while no signal could be observed in that without cells. We also can see from Figure 6 that the current decreased rapidly with an injection of 500 unit·mL⁻¹ catalase. As catalase is a selective scavenger of H₂O₂, it can be concluded that the increased current in the curve “with cells” was assigned to the reduction of H₂O₂.

4. Conclusions

In summary, a highly sensitive and selective enzyme-less sensor of H₂O₂ was successfully constructed with Ag-mSiO₂ NPs/GCE, enabling good electrocatalytic performance toward H₂O₂ reduction. The modified electrode exhibited a fast response to H₂O₂ concentration variation at an optimized working potential of −0.5 V. A wide linear range from 4 μM to 10 mM with a low LOD of 3 μM was obtained. In addition, the presented sensor showed high selectivity over commonly interfering substances, such as uric acid (UA), ascorbic acid (AA), and glucose. Furthermore, the Ag-mSiO₂ NPs/GCE based enzyme-less sensor exhibits good reproducibility and long-term stability. Accordingly, the constructed biosensor could be successfully used for the determination of H₂O₂ released from live cells. As ROS or H₂O₂ has been associated with cell cycle arrest, apoptosis, migration, and inflammation, or the antibacterial activity of certain bacteria such as Lactobacilli, our sensor has potential to help understand the role of H₂O₂ that plays in the biological and pathological systems. However, for monitoring the ROS or H₂O₂ from real samples, the stability of the constructed sensor still needs to be improved with employing more advanced nanomaterials because there is a lot of interferent in real clinical samples.