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Iodophenol blue-enhanced luminol chemiluminescence and its application to hydrogen peroxide and glucose detection
Author’s Accepted Manuscript Iodophenol blue-enhanced luminol chemiluminescence and its application to Hydrogen peroxide and glucose detection Dalong Yu, Ping Wang, Yanjun Zhao, Aiping Fan www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(15)30100-4 http://dx.doi.org/10.1016/j.talanta.2015.06.059 TAL15736
To appear in: Talanta Received date: 15 April 2015 Revised date: 16 June 2015 Accepted date: 20 June 2015 Cite this article as: Dalong Yu, Ping Wang, Yanjun Zhao and Aiping Fan, Iodophenol blue-enhanced luminol chemiluminescence and its application to Hydrogen peroxide and glucose detection, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.06.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Iodophenol blue-enhanced luminol chemiluminescence and its application to hydrogen peroxide and glucose detection
Dalong Yu, Ping Wang, Yanjun Zhao, Aiping Fan*
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, People’s Republic of China
*Corresponding author. Tel.: +86-22-27401191; Fax: +86-22-27409967. E-mail address: [email protected]
Abstract In this study, we found that iodophenol blue can enhance the week chemiluminescence (CL) of luminol-H2O2 system. With the aid of CL spectral, electron spin resonance (ESR) spectral measurements and studies on the effects of various free radical scavengers on the iodophenol blue-enhanced luminol-H2O2 system, we speculated that iodophenol blue may react with H2O2 and oxygen to produce oxidizing radical species such as OH and O2- resulting the formation of
1
O2. The generated
1
O2 may react with luminol anion
generating an unstable endoperoxide and subsequent 3-aminophthalate* (3-APA*). When the excited-state 3-APA returned to the ground-state, an enhanced CL was observed. Based on the H2O2 concentration dependence of the catalytic activity of iodophenol blue, a cheap, simple, sensitive CL assay 1
for the determination of H 2O2 was established. Under the optimum experimental conditions, a linear relationship between the relative CL intensity and H2O2 concentration in the range of 0.025-10 μM was obtained. As low as 14 nM H 2O2 can be sensitively detected by using the proposed method. The relative standard deviation for 5, 1 and 0.25 μM H 2O2 was 2.58%, 5.16% and 4.66%, respectively. By combining the glucose oxidase (GOx)-catalyzed oxidation reaction, CL detection of glucose was realized. The linear range of glucose detection was 0.1-30 μM with a detection limit of 0.06 μM. The proposed method has been applied to the detection of glucose in diluted serum.
Keywords: Chemiluminescence; Luminol; Hydrogen peroxide; Glucose; Iodophenol blue
1. Introduction Owing to its attracting features such as high sensitivity, low background signal, and simple instrumentation, CL has been exploited with a wide range of applications in different fields such as biotechnology, pharmaceutical analysis, molecular biology, clinical diagnosis, food analysis, and environmental analysis [1-5]. Luminol-H2O2 CL reaction, as a popular CL reaction, has been widely applied for the determination of various substances [6-8]. Several kinds of catalysts including enzymes [9], metal ions [10], and nanoparticles [11-16] were reported as effective catalyst of luminol-H2O2 CL reaction. Among them, horseradish peroxidase (HRP) is the most commonly used catalyst in luminol-H2O2 CL reaction, and has been widely used for the detection of hydrogen peroxide and other compounds through coupled enzymatic reactions.
2
However, the natural enzyme suffers from some drawbacks such as short shelf life, isolating difficulty, and high cost. In recent years, several kinds of nanoparticles including Au NPs [12], CuO NPs [13], Co3O4 NPs [14], Au nanocluster [15], Pt NPs [16], etc. were reported to give peroxidase-mimic properties and to catalyze the luminol-H2O2 reaction to emit light. In comparison with HRP, peroxidase-mimic nanoparticles are low-cost and less vulnerable to denaturation, and exhibit great promise in constructing CL determination schemes. However, the catalytical activity of nanoparticles in luminol CL reaction always depends on particle size and distribution. The preparation of nanoparticles with uniform size is still a big challenge. In addition, the toxicity of most nanoparticles is unkown. Hence, the discovery of molecular catalyst-enhanced system with both sensitivity and selectivity is still essential. In the present study, iodophenol blue which is a chemical indicator was found for the first time having catalytic activity on luminol-H2O2 CL reaction. To the best of our knowledge, the application of chemical indicators as catalyst for luminol-H2O2 CL system has not yet been reported. The structure of iodophenol blue is given in Fig. 1. In previous, certain chemical indicators, such as phenolphtha-lein, cresolphthalein, phenol red, bromophenol blue, etc. have been reported as enhancers of luminol-H2O2-HRP CL reaction [17-19]. In our preliminary experiment, the enhancing effect of iodophenol blue on luminol-H2O2-HRP CL system was firstly investigated. But it was found interestingly that blank signal (CL signal in the absence of HRP) of the luminol-H2O2-HRP-iodophenol blue system was very high indicating that iodophenol blue may catalyze luminol-H2O2 CL reaction directly even in the absence of HRP. In the present study, the catalytic mechanism of iodophenol in luminol-H2O2 system was investigated. And based on the H2O2 concentration
3
dependence of the catalytic activity of iodophenol blue, a sensitive and nonenzymatic CL method for H2O2 was then developed. Monitoring of blood glucose levels is extremely essential for diabetes who suffers from dysfunction of glucose uptake caused by insulin deficiency or resistance [20]. A large number of analytical methods for blood glucose including spectrophotometry [21-23], fluorimetry [24-26], amperometry [27,28] and chemiluminescence (CL) [29,30] have been developed. Because H2O2 is produced when glucose reacts with GOx in the presence of O2, the proposed method was applied for CL detection of glucose by combining the GOx-catalyzed oxidation reaction. The results indicated that this method is simple, cheap, and highly sensitive and selective for glucose detection, and can be used in diluted serum samples. (Figure 1) 2. Experimental 2.1. Chemicals. All chemicals and reagents were of analytical grade and used as received. Ultra pure water (18.2 MΩ cm-1) was used throughout the current work. Iodophenol blue was obtained from Tianjin Heowns Biochemical Technology Co., Ltd. (China). Luminol was purchased from Alfa Aesar (Tianjin, China). Glucose oxidase (GOx, 100-250 U mg-1) was purchased from Shanghai Yuanye Biological Technology Co. Ltd.
(China).
5,5-Dimethyl-1-pyrroline
N-oxide
(DMPO)
and
2,2,6,6-tetramethyl-4-piperidone (TEMP) were purchased from J&K Scientific Ltd. (Beijing, China). Hydrogen peroxide (30%, v/v) and other chemical reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).
4
2.2. Apparatus. CL
measurements
and
CL
spectra
were
performed
with
a
BPCL
chemiluminescence analyzer (Beijing, China) with a series of high-energy optical filters of 230, 260, 290, 320, 350, 380, 400, 425, 440, 460, 490, 535, 555, 575, 620, and 640 nm between the CL measuring cup holder and PMT. 2.3. Preparation and reagents A stock solution of iodophenol blue was prepared at a 0.5 M concentration in dimethyl formamide and the working solution of iodophenol blue (0.25 mM) was made by gradually diluting relative stock solution with 0.1 M HAc-KAc buffer solution (pH 6.5). Stock solution for 0.01 M luminol was made in 0.01 M NaOH solution and then this solution was diluted with 0.1 M Tris buffer solution (pH 10.0) to prepare a 0.5 mM working solution of luminol. Working solutions of H2O2 were prepared fresh daily by dilution of 30% (v/v) H2O2. 2.4. Assay procedure of CL detection of H2O2 For CL detection of H2O2, 40 μL of different concentrations of H2O2 were mixed with 10 μL of 0.25 mM iodophenol blue in a 14 Í 40 mm glass tube at first, and 50 μL of 0.5 mM luminol was then injected into the glass tube immediately. At the same time, the light emission was measured by the BPCL chemiluminescence analyzer without the use of optical filter. The CL profile and intensity were displayed and integrated for a 0.1 s interval at -1000 V. 2.5. Method validation for CL detection of H2O2 To test the linearity and range for CL detection of H2O2, 40 μL of 0, 0.025, 0.25, 0.5, 1, 2.5, 5, and 10 μM H2O2 was mixed with 10 μL of 0.25 mM iodophenol blue, 5
respectively. The CL signals were then detected as described in “assay procedure of CL detection of H2O2”. The precision of CL intensity for H2O2 detection was determinated by sequentially analyzing sets of several H2O2 standard solution of different concentration (0.25, 1, and 5 μM). Each concentration level of H2O2 was measured eight times parallelly. To evaluate the sepecificity of the proposed CL method for H2O2 detection, the effect of interferences including Na+, K+, Ag+, NH4+, Ca2+, Mg2+, Co2+, Cu2+, Mn2+, Fe3+, Cl-, Ac-, HCO3-, H2PO4-, NO3- and SO42- on CL intensity was investigated, respectively. 40 μL of 5 μM H2O2 without interferent and with increasing concentration (from 5 μM to 1 mM) of interferent was mixed with 10 μL of 0.25 mM iodophenol blue, respectively. The CL signals were then detected as described in “assay procedure of CL detection of H 2O2”. 2.6. Assay procedure of CL detection of glucose For CL detection of glucose, 40 μL of reaction solution containing 2.5 mM phosphate buffer (pH 7.4), 0.22 U GOx and different concentrations of glucose were incubated at 37 ◦C for 10 min. Then, 10 μL of 0.25 mM iodophenol blue was added into the above reaction solution. The result mixture was then transferred into a 14 Í 40 mm glass tube, and 50 μL of 0.5 mM luminol was injected into the glass tube immediately for CL detection as described in “assay procedure of CL detection of H2O2”. The concentration of glucose was quantified by CL intensity. 2.7. Method validation for CL detection of glucose To test the linearity and range for CL detection of glucose, 0, 0.1, 1, 10, 20, 30 μM glucose was employed, and the CL signals were detected as described in “assay procedure of CL detection of glucose”. To determine the specificity of the CL glucose
6
detection system, PBS buffer and 10 μM D-lactose, D-xylose and D-mannose (dissolved in 2.5 mM phosphate buffer) reacted with 0.22 U GOx at 37 ◦C for 10 min. The CL signal of the resulting mixture was then measured as described above. 3. Results and discussion 3.1. Enhancement of luminol-H2O2 reaction by iodophenol blue The effects of iodophenol blue on the luminol-H2O2 CL system were investigated. Fig. 2A shows the kinetic curves of luminol-H2O2 CL system in the presence and in the absence of iodophenol blue, respectively. In the absence of iodophenol blue, the oxidation of luminol by H2O2 in alkaline solution is a relatively slow reaction process and the CL intensity is weak. With the addition of iodophenol blue, a fast and intense CL was observed indicating that iodophenol blue could greatly enhance the luminol-H2O2 CL reaction. The CL spectra were measured using a series of high-energy cutoff filters of 230, 260, 290, 320, 350, 380, 400, 425, 440, 460, 490, 535, 555, 575, 620, and 640 nm. Fig. 2B displays the CL spectra of luminol-H2O2 system in either the absence or the presence of iodophenol blue. The spectra reveal that the maximal emission in both cases is 425 nm, indicating that the luminophors are still the excited state 3-aminophthalate anions (3-APA*). (Figure 2) 3.2. Mechanism Discussion In order to investigate the mechanism of the CL-enhancing phenomena, several specific quenchers were added into the reaction solution. We found that 1O2 plays an important role in the above CL system, which could be identified by using specific quencher for 1O2 such as sodium azide (NaN3). 91.2% CL intensity was inhibited by
7
addition of 0.1 M NaN3 indicating 1O2 was involved in the luminol-H2O2-iodophenol blue CL system. This speculation was further confirmed by using room temperature ESR spectra [31]. TEMP, a specific target molecule of 1O2, was used for capturing 1
O2 resulting in the formation of the adduct 2,2,6,6-tetramethyl-4-piperidine-N-oxide
(TEMPO) which has a characteristic spectrum of nitroxide radical. Fig. 3 shows the room temperature ESR spectra of the luminol-H2O2 CL system in the absence and presence of iodophenol blue, respectively. The existence of 0.5 mM iodophenol blue in the reaction mixture can enhance the intensity of the characteristic signal of TEMPO radical obviously indicating catalytic effect of iodophenol blue on the formation of 1O2. (Figure 3) Besides
the
important
observation
of
1
O2-dependent
CL
in
the
luminol-H2O2-iodophenol blue system, the generation of other oxidizing radical species appeared to be essential in the CL reaction. β-carotene is generally used as broad-spectrum radical scavenger. However, And 80.3% CL intensity was inhibited by addition of 1 mg/mL β-carotene. In our experiment, it is possible that iodophenol blue acts as catalyst for the decomposition of H 2O2 to generate hydroxyl radical (OH ). This speculation was comfirmed by addition of methanol which is generally used as a hydroxyl radical scavenger to the reaction solution. And 71.2% CL intensity was inhibited by addition of 5% methanol revealing that OH, an important intermediate product in luminol CL reaction, also formed in the luminol-H2O2-iodophenol blue system. The formation of OH during the CL reaction was further comfirmed by ESR measurements with the addition of the spin trapping agent DMPO which is a specific target molecule of OH. As shown in Fig. 4, a typical ESR spectrum for DMPO-OH adducts was observed in the 8
presence of iodophenol blue, while there was no such signal in the absence of iodophenol blue. The results indicated the existence of OH during the catalytic oxidation process. Superoxide anion (O2-) may also play an important role in the catalytic oxidation process. 89.5% CL intensity was inhibited by addition of 10 U super oxygen dehydrogenises (SOD), which is generally used as superoxide anion (O2-) scavenger. We speculated that dissolved oxygen may play an important role for the generation of O2-, because about 90% CL intensity was inhibited when the reaction solutions was bubbled with N 2 for 30 min before the CL reaction. (Figure 4) In view of the above results, it could be speculated that iodophenol blue may react with H2O2 and oxygen to produce oxidizing radical species such as OH and O2-. And the interaction between these reactive oxygen species may induce the generation of 1O2 according to previous reports [32]. Then, the generated 1O2 reacted with luminol anion generating an unstable endoperoxide and subsequent 3-aminophthalate* (3-APA*). When the exited-state 3-APA returned to the ground-state, an enhanced CL was observed. 3.3. CL detection of H2O2 Based on the novel luminol-H2O2-iodophenol blue CL system, a sensitive and simple hydrogen peroxide detection method was developed (Fig. 5). With increasing concentration of H2O2, gradually increased CL signals were obtained, indicating the feasibility of H2O2 determination by using the above novel CL system. (Figure 5)
9
To establish a highly sensitive and rapid determination of H2O2, assay conditions including the pH of Tris buffer for the dilution of luminol, the pH of HAc-KAc buffer for the dilution of iodophenol blue, and the concentrations of luminol and iodophenol blue were studied respectively. The effect of luminol concentration on the CL intensity was studied in the range of 0.01-1 mM (Fig. 6A). It was found that the relative CL intensity (refers to sample signal minus blank) increased with increasing luminol concentration in the range of 0.01-0.5 mM, and above 0.5 mM luminol, the relative CL intensity decreased which is attributed to a significantly enhanced background. Hence, 0.5 mM luminal was selected for the following studies. The efficiency of luminol CL reaction is dependent on the pH of the reaction medium. The effect of the pH of luminol solution on the CL intensity was studied using Tris buffer. The pH of Tris buffer was adjusted with 0.1 M HCl or NaOH. It was found that there was no obvious CL signal when the pH was less than 9.0, and strong CL emission was observed when the pH scale is from 10.0 to 12.0. As shown in Fig. 6B, both sample signal and blank monotonously increased with the pH value over the whole tested range. The signal to noise ratio was almost constant between pH 10.0 and pH 12.0. Therefore, the optimal pH of Tris buffer for the dilution of luminol was set at 10. HAc-KAc buffer was used for the dilution of iodophenol blue. The pH effect of HAc-KAc buffer on CL intensity was studied. It was found in our preliminary experiment that the CL signal of the luminol-H2O2-iodophenol blue CL system was not stable when iodophenol blue was diluted in HAc-KAc buffer with pH lower than 5.5. So the pH of HAc-KAc buffer in the range 5.5-7.5 was investigated. In principle,
10
the pH of iodophenol blue solution affects the pH of the final reaction solution which affects the efficiency of luminol CL reaction. Therefore, higher relative CL intensities were demonstrated in the higher pH range of the buffer (Fig. 6C). The relative CL intensity increased when the pH of HAc-KAc buffer was increased from 5.5 to 6.5, and then maintained almost the same in the range of 6.5 to 7.5. Hence, the optimal pH of HAc-KAc for the dilution of iodophenol blue was selected at 6.5. And the final pH of the reaction solution was 8.87 under the optimal pH conditions. Finally, influence of the concentrations of iodophenol blue on the CL intensity was examined in the range of 0.01-1 mM. It was found that both signal and blank was also increased with the rise of iodophenol blue concentration. However, the signal to noise ratio increased when the concentration of iodophenol blue was increased from 0.01 to 0.1 mM, and then maintained almost the same in the range of 0.1-0.25 mM iodophenol blue (Fig. 6D). After that, the signal to noise ratio was decreased gradually. Hence, 0.25 mM iodophenol blue was selected for the following experiments. (Figure 6) 3.4. Assay performance for H 2O2 detection Under the optimum experimental conditions, the relative CL intensity of iodophenol blue-catalyzed CL system was linearly dependent on the concentration of H2O2 in the range 0.025-10 μM (Fig. 7). The regression equation for the signal I (relative CL intensity) was I=827.08+2979C (H2O2) [μM], with a correlation coefficient 0.997 (n=3). The LOD and LOQ was defined as the concentration of H2O2 which gave a CL intensity 3× and 10× the standard deviation of a blank (which was detected eight times parallelly). LOD (3 σ/s) and LOQ (10 σ/s) values 11
were found to be 0.014 and 0.022 μM, respectively. In addition, a series of 8 repetitive measurements of 5, 1 and 0.25 μM H2O2 were employed for estimating the precision. The relative standard deviation was 2.58%, 5.16% and 4.66% for 5, 1 and 0.25 μM H2O2, respectively. (Figure 7) In order to investigate the selectivity of the proposed CL method, varying amounts of possible interferents were added to solution of 5 μM H2O2 and the CL signal was recorded. The tolerable concentration of each possible interferent was taken as a 5% relative error in the CL signal. The influence of ions to 5 μM H2O2 was first evaluated in the absence of EDTA. The results were listed in Table 1. The most serious interference was observed with Fe ions. EDTA, as a chelate reagent, was then added to sample solution to eliminate the interference derived from Fe ions. The experimental results demonstrated that the influence of Fe ions is highly diminished by the addition of 0.0001 M EDTA. The recovery of Fe ions in the presence of 0.0001 M EDTA was 96.32% indicating that the addition of EDTA to sample solution should be advantageous. (Table 1) 3.5. Assay performance for glucose detection Hydrogen peroxide is the main product of GOx-catalyzed reaction. Therefore, the proposed CL method was applied for CL detection of glucose. The CL detection of glucose can be performed in two steps: 1) GOx catalyzes the reaction between glucose and oxygen resulting in the formation of gluconic acid and H 2O2; 2) the generated H2O2 reacts with iodophenol blue and luminol to produce light.
12
The detailed procedure is described in the experimental section. Fig. 8A shows the linear range of glucose detected in PBS buffer which is from 0.1 to 30 μM with a detection limit of 0.06 μM. The regression equation for the signal I (relative CL intensity) was I=3385+2060C (glucose) [μM], with a correlation coefficient 0.989 (n=3). To evaluate the selectivity of the method, control experiments were performed using buffer solution, D-lactose, D-xylose, and D-mannose. A high CL response was observed when 1 μM of lucose was tested, while negligible signals were obtained using buffer solution, 1 μM of D-lactose, D-xylose, and D-mannose (Fig. 8B). The results show promising selectivity of the proposed CL assay for glucose. Finally, the proposed CL method was used for the determination of glucose in diluted human serum samples (Table 2). A validation of the method was carried out by the glucose paper method as a reference method whose results were also listed in Table 2. As shown in Table 2, there is a good agreement between the proposed CL method and the glucose paper method, indicating the capability of the proposed CL method being applied to the determination of glucose in biological sample with satisfactory results. (Figure 8) (Table 2)
4. Conclusion
In summary, iodophenol blue was found to catalyze the luminol-H2O2 CL reaction in this study. The luminophor of the iodophenol blue catalyzed luminol-H2O2 CL system was 3-APA*. The mechanism study showed that 13
iodophenol blue served as catalyst to accelerate the generation of oxidizing radical species such as OH and O2-, and subsequent formation of 1O2 which reacted with luminol anion to emit light. Based on the novel CL system, a simple, sensitive and selective CL assay for H 2O2 was developed. The proposed method demonstrated high sensitivity that can detect as low as 14 nM H 2O2. The proposed CL assay was applied for the determination of glucose in diluted human serum by combining with glucose oxide. The results of CL detection of glucose were in good agreement with those obtained by a commercial paper-based method. The results demonstrate the potential application of the proposed method in determination of enzyme-coupled catalyzed biologically important molecules, such as uric acid, cholestrerol, and lactic acid. Table 3 compared the metrological parameters and green aspects of the proposed method with reported methods indicating that the proposed method is simple, sensitive, and applicable. This work not only displays a new property of iodophenol blue in CL field and enriches luminol CL mechanism but also shows great potential applications in analytical and biochemical fields.
(Table 3)
Acknowledgment
This study was supported by National Natural Science Foundation of China (21475094) and the 973 project (nos. 2015CB856500). Reference
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Fig. 3 ESR spectra of nitroxide radicals generated by the reaction of TEMP probe with 1O2. Conditions: modulation amplitude, 1.00 G; microwave power, 20.000 mW; receiver gain, 5.02e + 003; sweep width, 150 G. The ESR measurements were achieved with a Bruker A300 spectrometer operating in the X-band at room temperature. Fig. 4 ESR spectra of DMPO-OH adduct in the luminal-H2O2 system in the presence or absence of iodophenol blue. Condition: modulation amplitude, 1.00 G; microwave power, 20.020 mW; receiver gain, 5.02e + 005; sweep width, 200.000 G. The ESR measurements were achieved with a Bruker EMX spectrometer operating in the X-band at room temperature. Fig. 5. The Kinetic characteristics of the lumimol-H2O2-iodophenol blue system. Experimental conditions were the same as Fig. 2A. Fig. 6 Effects of the reactant conditions on luminol-H2O2 CL system in the presence of iodophenol blue. (A) Effect of luminol concentration on relative CL intensity. Experimental conditions: iodophenol blue in 0.1 M HAc-KAc buffer (pH 6.5), 10 μL; 5 μM H2O2, 40 μL; different concentrations of luminol in 0.1 M Tris buffer (pH 10), 50 μL. (B) Effect of Tris buffer pH on CL signal. Experimental conditions: 0.5 mM luminol in 0.1 M Tris buffer (PH 8-12), 50 μL; other experimental conditions were the same as Fig. 6A. (C) Effect of HAc-KAc buffer pH on CL signal. Experimental conditions: 0.1 mM iodophenol blue in 0.1 M HAc-KAc buffer solution (PH 5.5-7.5), 10 μL; other experimental conditions were the same as Fig. 6B. (D) Effect of
18
iodophenol blue concentration on CL signal. Experimental conditions: different concentrations of iodophenols blue in 0.1 M HAc-KAc buffer (pH 6.5), other experimental conditions were the same as Fig. 6C. Fig. 7 Calibration curve of CL intensity vs concentration of H2O2. The inset shows the CL responses of H2O2 detection at low concentrations. Experimental conditions: 0.25 mM iodophenol blue in 0.1 M HAc-KAc buffer (pH 6.5), 10 μL; different concentrations of H2O2, 40 μL; 0.5 mM luminol in 0.1 M Tris buffer (pH 10), 50 μL. Fig. 8 (A) CL response of the luminol-glucose/GOx-iodophenol blue system in the prensence of different concentrations of glucose. Experimental conditions: luminol, 0.5 mM; iodophenol blue, 0.25 mM; GOx, 10U; glucose, 0.1, 0.5, 1, 10, 20, 30 μM. (B) Selectivity of the sensing for glucose compared to other possible interfering targets. The concentration of glucose, PBS, D-lactose, D-xylose and D-mannose is 10 μM. Other experimental conditions were the same as denoted in Fig. 8 (A). Table 1 Recoveries of 5 μM H2O2 in the presence of interferents Coexisting species
Concentration (mg L-1)
Recovery (%)
Na+
23
101.13
Mn2+
27.5
95.67
K+
19.5
103.47
Fe3+
28
40.37
Ag+
54
98.66
Cl-
36
98.58
NH4+
18
100.66
Ac-
22
103.47
Ca2+
20
99.13
HCO3-
30
97.88
Mg2+
12
99.62
H2PO4-
48
98.67
Co2+
29.5
98.58
NO3-
31
98.66
19
Coexisting Concentration Recovery (mg L-1) species (%)
Cu2+
32
SO42-
98.53
48
100.66
Table 2 Results of determination of glucose in diluted serum Proposed method1)
Glucose paper method
/mmol L-1
/mmol L-1
1
3.01 ± 0.41
2.9
2
5.63 ± 0.46
6.0
3
2.01 ± 0.17
2.3
4
7.51 ± 0.51
8.6
5
10.20 ± 1.63
9.7
Sample
1)
± Expanded uncertainty (U) for k = 2
Table 3 Comparision of sensitivity for different H2O2 assay methods H2O2 detection
Drawbacks
Analytical method
Detection
Application
Linearity
A
B
Ĝ
limit
range
Electrochemistry[29]
32.6 μM
80.0-372.0 mM
-
Ĝ
Electrochemistry [30]
1.1 μM
10.0-1900 μM
H2O2 in human urine
Ĝ
Electrochemistry [31]
10 nM
0.1-70 μM
-
Ĝ
CL [32]
0.35 μM
1.0-4000 μM
-
CL [10]
11 nM
0.1-5.0 μM
-
CL
14 nM
this study
glucose in human serum
20
Ĝ
C
Phosphorescence [33]
140 nM
1.0-60 μM
glucose in blood and saliva
Fluorimetry [22]
0.34 μM
5.0-200 μM
L-lactate in human serum
Amperometry [34]
0.05 μM
0.1-120 μM
H2O2 in beverages
Ĝ
Ĝ
A represents complex modification or synthesis processes are involved. B represents use of toxic organic reagent or toxicity unknown nanoparticles. C represents a time-consuming detection procedure was needed.
Highlights 1. A novel luminol CL system was developed and applied to the detection of hydrogen peroxide. 2. The working range is 0.025-10 μM and the detection limit is 14 nM. 3. The new CL assay exhibits high sensitivity and extraordinary specificity.
4. The proposed method has been applied to the detection of glucose in diluted serum.
21
Ĝ
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
*Graphical Abstract (for review)
PII: DOI: Reference:
S0039-9140(15)30100-4 http://dx.doi.org/10.1016/j.talanta.2015.06.059 TAL15736
To appear in: Talanta Received date: 15 April 2015 Revised date: 16 June 2015 Accepted date: 20 June 2015 Cite this article as: Dalong Yu, Ping Wang, Yanjun Zhao and Aiping Fan, Iodophenol blue-enhanced luminol chemiluminescence and its application to Hydrogen peroxide and glucose detection, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.06.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Iodophenol blue-enhanced luminol chemiluminescence and its application to hydrogen peroxide and glucose detection
Dalong Yu, Ping Wang, Yanjun Zhao, Aiping Fan*
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, People’s Republic of China
*Corresponding author. Tel.: +86-22-27401191; Fax: +86-22-27409967. E-mail address: [email protected]
Abstract In this study, we found that iodophenol blue can enhance the week chemiluminescence (CL) of luminol-H2O2 system. With the aid of CL spectral, electron spin resonance (ESR) spectral measurements and studies on the effects of various free radical scavengers on the iodophenol blue-enhanced luminol-H2O2 system, we speculated that iodophenol blue may react with H2O2 and oxygen to produce oxidizing radical species such as OH and O2- resulting the formation of
1
O2. The generated
1
O2 may react with luminol anion
generating an unstable endoperoxide and subsequent 3-aminophthalate* (3-APA*). When the excited-state 3-APA returned to the ground-state, an enhanced CL was observed. Based on the H2O2 concentration dependence of the catalytic activity of iodophenol blue, a cheap, simple, sensitive CL assay 1
for the determination of H 2O2 was established. Under the optimum experimental conditions, a linear relationship between the relative CL intensity and H2O2 concentration in the range of 0.025-10 μM was obtained. As low as 14 nM H 2O2 can be sensitively detected by using the proposed method. The relative standard deviation for 5, 1 and 0.25 μM H 2O2 was 2.58%, 5.16% and 4.66%, respectively. By combining the glucose oxidase (GOx)-catalyzed oxidation reaction, CL detection of glucose was realized. The linear range of glucose detection was 0.1-30 μM with a detection limit of 0.06 μM. The proposed method has been applied to the detection of glucose in diluted serum.
Keywords: Chemiluminescence; Luminol; Hydrogen peroxide; Glucose; Iodophenol blue
1. Introduction Owing to its attracting features such as high sensitivity, low background signal, and simple instrumentation, CL has been exploited with a wide range of applications in different fields such as biotechnology, pharmaceutical analysis, molecular biology, clinical diagnosis, food analysis, and environmental analysis [1-5]. Luminol-H2O2 CL reaction, as a popular CL reaction, has been widely applied for the determination of various substances [6-8]. Several kinds of catalysts including enzymes [9], metal ions [10], and nanoparticles [11-16] were reported as effective catalyst of luminol-H2O2 CL reaction. Among them, horseradish peroxidase (HRP) is the most commonly used catalyst in luminol-H2O2 CL reaction, and has been widely used for the detection of hydrogen peroxide and other compounds through coupled enzymatic reactions.
2
However, the natural enzyme suffers from some drawbacks such as short shelf life, isolating difficulty, and high cost. In recent years, several kinds of nanoparticles including Au NPs [12], CuO NPs [13], Co3O4 NPs [14], Au nanocluster [15], Pt NPs [16], etc. were reported to give peroxidase-mimic properties and to catalyze the luminol-H2O2 reaction to emit light. In comparison with HRP, peroxidase-mimic nanoparticles are low-cost and less vulnerable to denaturation, and exhibit great promise in constructing CL determination schemes. However, the catalytical activity of nanoparticles in luminol CL reaction always depends on particle size and distribution. The preparation of nanoparticles with uniform size is still a big challenge. In addition, the toxicity of most nanoparticles is unkown. Hence, the discovery of molecular catalyst-enhanced system with both sensitivity and selectivity is still essential. In the present study, iodophenol blue which is a chemical indicator was found for the first time having catalytic activity on luminol-H2O2 CL reaction. To the best of our knowledge, the application of chemical indicators as catalyst for luminol-H2O2 CL system has not yet been reported. The structure of iodophenol blue is given in Fig. 1. In previous, certain chemical indicators, such as phenolphtha-lein, cresolphthalein, phenol red, bromophenol blue, etc. have been reported as enhancers of luminol-H2O2-HRP CL reaction [17-19]. In our preliminary experiment, the enhancing effect of iodophenol blue on luminol-H2O2-HRP CL system was firstly investigated. But it was found interestingly that blank signal (CL signal in the absence of HRP) of the luminol-H2O2-HRP-iodophenol blue system was very high indicating that iodophenol blue may catalyze luminol-H2O2 CL reaction directly even in the absence of HRP. In the present study, the catalytic mechanism of iodophenol in luminol-H2O2 system was investigated. And based on the H2O2 concentration
3
dependence of the catalytic activity of iodophenol blue, a sensitive and nonenzymatic CL method for H2O2 was then developed. Monitoring of blood glucose levels is extremely essential for diabetes who suffers from dysfunction of glucose uptake caused by insulin deficiency or resistance [20]. A large number of analytical methods for blood glucose including spectrophotometry [21-23], fluorimetry [24-26], amperometry [27,28] and chemiluminescence (CL) [29,30] have been developed. Because H2O2 is produced when glucose reacts with GOx in the presence of O2, the proposed method was applied for CL detection of glucose by combining the GOx-catalyzed oxidation reaction. The results indicated that this method is simple, cheap, and highly sensitive and selective for glucose detection, and can be used in diluted serum samples. (Figure 1) 2. Experimental 2.1. Chemicals. All chemicals and reagents were of analytical grade and used as received. Ultra pure water (18.2 MΩ cm-1) was used throughout the current work. Iodophenol blue was obtained from Tianjin Heowns Biochemical Technology Co., Ltd. (China). Luminol was purchased from Alfa Aesar (Tianjin, China). Glucose oxidase (GOx, 100-250 U mg-1) was purchased from Shanghai Yuanye Biological Technology Co. Ltd.
(China).
5,5-Dimethyl-1-pyrroline
N-oxide
(DMPO)
and
2,2,6,6-tetramethyl-4-piperidone (TEMP) were purchased from J&K Scientific Ltd. (Beijing, China). Hydrogen peroxide (30%, v/v) and other chemical reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).
4
2.2. Apparatus. CL
measurements
and
CL
spectra
were
performed
with
a
BPCL
chemiluminescence analyzer (Beijing, China) with a series of high-energy optical filters of 230, 260, 290, 320, 350, 380, 400, 425, 440, 460, 490, 535, 555, 575, 620, and 640 nm between the CL measuring cup holder and PMT. 2.3. Preparation and reagents A stock solution of iodophenol blue was prepared at a 0.5 M concentration in dimethyl formamide and the working solution of iodophenol blue (0.25 mM) was made by gradually diluting relative stock solution with 0.1 M HAc-KAc buffer solution (pH 6.5). Stock solution for 0.01 M luminol was made in 0.01 M NaOH solution and then this solution was diluted with 0.1 M Tris buffer solution (pH 10.0) to prepare a 0.5 mM working solution of luminol. Working solutions of H2O2 were prepared fresh daily by dilution of 30% (v/v) H2O2. 2.4. Assay procedure of CL detection of H2O2 For CL detection of H2O2, 40 μL of different concentrations of H2O2 were mixed with 10 μL of 0.25 mM iodophenol blue in a 14 Í 40 mm glass tube at first, and 50 μL of 0.5 mM luminol was then injected into the glass tube immediately. At the same time, the light emission was measured by the BPCL chemiluminescence analyzer without the use of optical filter. The CL profile and intensity were displayed and integrated for a 0.1 s interval at -1000 V. 2.5. Method validation for CL detection of H2O2 To test the linearity and range for CL detection of H2O2, 40 μL of 0, 0.025, 0.25, 0.5, 1, 2.5, 5, and 10 μM H2O2 was mixed with 10 μL of 0.25 mM iodophenol blue, 5
respectively. The CL signals were then detected as described in “assay procedure of CL detection of H2O2”. The precision of CL intensity for H2O2 detection was determinated by sequentially analyzing sets of several H2O2 standard solution of different concentration (0.25, 1, and 5 μM). Each concentration level of H2O2 was measured eight times parallelly. To evaluate the sepecificity of the proposed CL method for H2O2 detection, the effect of interferences including Na+, K+, Ag+, NH4+, Ca2+, Mg2+, Co2+, Cu2+, Mn2+, Fe3+, Cl-, Ac-, HCO3-, H2PO4-, NO3- and SO42- on CL intensity was investigated, respectively. 40 μL of 5 μM H2O2 without interferent and with increasing concentration (from 5 μM to 1 mM) of interferent was mixed with 10 μL of 0.25 mM iodophenol blue, respectively. The CL signals were then detected as described in “assay procedure of CL detection of H 2O2”. 2.6. Assay procedure of CL detection of glucose For CL detection of glucose, 40 μL of reaction solution containing 2.5 mM phosphate buffer (pH 7.4), 0.22 U GOx and different concentrations of glucose were incubated at 37 ◦C for 10 min. Then, 10 μL of 0.25 mM iodophenol blue was added into the above reaction solution. The result mixture was then transferred into a 14 Í 40 mm glass tube, and 50 μL of 0.5 mM luminol was injected into the glass tube immediately for CL detection as described in “assay procedure of CL detection of H2O2”. The concentration of glucose was quantified by CL intensity. 2.7. Method validation for CL detection of glucose To test the linearity and range for CL detection of glucose, 0, 0.1, 1, 10, 20, 30 μM glucose was employed, and the CL signals were detected as described in “assay procedure of CL detection of glucose”. To determine the specificity of the CL glucose
6
detection system, PBS buffer and 10 μM D-lactose, D-xylose and D-mannose (dissolved in 2.5 mM phosphate buffer) reacted with 0.22 U GOx at 37 ◦C for 10 min. The CL signal of the resulting mixture was then measured as described above. 3. Results and discussion 3.1. Enhancement of luminol-H2O2 reaction by iodophenol blue The effects of iodophenol blue on the luminol-H2O2 CL system were investigated. Fig. 2A shows the kinetic curves of luminol-H2O2 CL system in the presence and in the absence of iodophenol blue, respectively. In the absence of iodophenol blue, the oxidation of luminol by H2O2 in alkaline solution is a relatively slow reaction process and the CL intensity is weak. With the addition of iodophenol blue, a fast and intense CL was observed indicating that iodophenol blue could greatly enhance the luminol-H2O2 CL reaction. The CL spectra were measured using a series of high-energy cutoff filters of 230, 260, 290, 320, 350, 380, 400, 425, 440, 460, 490, 535, 555, 575, 620, and 640 nm. Fig. 2B displays the CL spectra of luminol-H2O2 system in either the absence or the presence of iodophenol blue. The spectra reveal that the maximal emission in both cases is 425 nm, indicating that the luminophors are still the excited state 3-aminophthalate anions (3-APA*). (Figure 2) 3.2. Mechanism Discussion In order to investigate the mechanism of the CL-enhancing phenomena, several specific quenchers were added into the reaction solution. We found that 1O2 plays an important role in the above CL system, which could be identified by using specific quencher for 1O2 such as sodium azide (NaN3). 91.2% CL intensity was inhibited by
7
addition of 0.1 M NaN3 indicating 1O2 was involved in the luminol-H2O2-iodophenol blue CL system. This speculation was further confirmed by using room temperature ESR spectra [31]. TEMP, a specific target molecule of 1O2, was used for capturing 1
O2 resulting in the formation of the adduct 2,2,6,6-tetramethyl-4-piperidine-N-oxide
(TEMPO) which has a characteristic spectrum of nitroxide radical. Fig. 3 shows the room temperature ESR spectra of the luminol-H2O2 CL system in the absence and presence of iodophenol blue, respectively. The existence of 0.5 mM iodophenol blue in the reaction mixture can enhance the intensity of the characteristic signal of TEMPO radical obviously indicating catalytic effect of iodophenol blue on the formation of 1O2. (Figure 3) Besides
the
important
observation
of
1
O2-dependent
CL
in
the
luminol-H2O2-iodophenol blue system, the generation of other oxidizing radical species appeared to be essential in the CL reaction. β-carotene is generally used as broad-spectrum radical scavenger. However, And 80.3% CL intensity was inhibited by addition of 1 mg/mL β-carotene. In our experiment, it is possible that iodophenol blue acts as catalyst for the decomposition of H 2O2 to generate hydroxyl radical (OH ). This speculation was comfirmed by addition of methanol which is generally used as a hydroxyl radical scavenger to the reaction solution. And 71.2% CL intensity was inhibited by addition of 5% methanol revealing that OH, an important intermediate product in luminol CL reaction, also formed in the luminol-H2O2-iodophenol blue system. The formation of OH during the CL reaction was further comfirmed by ESR measurements with the addition of the spin trapping agent DMPO which is a specific target molecule of OH. As shown in Fig. 4, a typical ESR spectrum for DMPO-OH adducts was observed in the 8
presence of iodophenol blue, while there was no such signal in the absence of iodophenol blue. The results indicated the existence of OH during the catalytic oxidation process. Superoxide anion (O2-) may also play an important role in the catalytic oxidation process. 89.5% CL intensity was inhibited by addition of 10 U super oxygen dehydrogenises (SOD), which is generally used as superoxide anion (O2-) scavenger. We speculated that dissolved oxygen may play an important role for the generation of O2-, because about 90% CL intensity was inhibited when the reaction solutions was bubbled with N 2 for 30 min before the CL reaction. (Figure 4) In view of the above results, it could be speculated that iodophenol blue may react with H2O2 and oxygen to produce oxidizing radical species such as OH and O2-. And the interaction between these reactive oxygen species may induce the generation of 1O2 according to previous reports [32]. Then, the generated 1O2 reacted with luminol anion generating an unstable endoperoxide and subsequent 3-aminophthalate* (3-APA*). When the exited-state 3-APA returned to the ground-state, an enhanced CL was observed. 3.3. CL detection of H2O2 Based on the novel luminol-H2O2-iodophenol blue CL system, a sensitive and simple hydrogen peroxide detection method was developed (Fig. 5). With increasing concentration of H2O2, gradually increased CL signals were obtained, indicating the feasibility of H2O2 determination by using the above novel CL system. (Figure 5)
9
To establish a highly sensitive and rapid determination of H2O2, assay conditions including the pH of Tris buffer for the dilution of luminol, the pH of HAc-KAc buffer for the dilution of iodophenol blue, and the concentrations of luminol and iodophenol blue were studied respectively. The effect of luminol concentration on the CL intensity was studied in the range of 0.01-1 mM (Fig. 6A). It was found that the relative CL intensity (refers to sample signal minus blank) increased with increasing luminol concentration in the range of 0.01-0.5 mM, and above 0.5 mM luminol, the relative CL intensity decreased which is attributed to a significantly enhanced background. Hence, 0.5 mM luminal was selected for the following studies. The efficiency of luminol CL reaction is dependent on the pH of the reaction medium. The effect of the pH of luminol solution on the CL intensity was studied using Tris buffer. The pH of Tris buffer was adjusted with 0.1 M HCl or NaOH. It was found that there was no obvious CL signal when the pH was less than 9.0, and strong CL emission was observed when the pH scale is from 10.0 to 12.0. As shown in Fig. 6B, both sample signal and blank monotonously increased with the pH value over the whole tested range. The signal to noise ratio was almost constant between pH 10.0 and pH 12.0. Therefore, the optimal pH of Tris buffer for the dilution of luminol was set at 10. HAc-KAc buffer was used for the dilution of iodophenol blue. The pH effect of HAc-KAc buffer on CL intensity was studied. It was found in our preliminary experiment that the CL signal of the luminol-H2O2-iodophenol blue CL system was not stable when iodophenol blue was diluted in HAc-KAc buffer with pH lower than 5.5. So the pH of HAc-KAc buffer in the range 5.5-7.5 was investigated. In principle,
10
the pH of iodophenol blue solution affects the pH of the final reaction solution which affects the efficiency of luminol CL reaction. Therefore, higher relative CL intensities were demonstrated in the higher pH range of the buffer (Fig. 6C). The relative CL intensity increased when the pH of HAc-KAc buffer was increased from 5.5 to 6.5, and then maintained almost the same in the range of 6.5 to 7.5. Hence, the optimal pH of HAc-KAc for the dilution of iodophenol blue was selected at 6.5. And the final pH of the reaction solution was 8.87 under the optimal pH conditions. Finally, influence of the concentrations of iodophenol blue on the CL intensity was examined in the range of 0.01-1 mM. It was found that both signal and blank was also increased with the rise of iodophenol blue concentration. However, the signal to noise ratio increased when the concentration of iodophenol blue was increased from 0.01 to 0.1 mM, and then maintained almost the same in the range of 0.1-0.25 mM iodophenol blue (Fig. 6D). After that, the signal to noise ratio was decreased gradually. Hence, 0.25 mM iodophenol blue was selected for the following experiments. (Figure 6) 3.4. Assay performance for H 2O2 detection Under the optimum experimental conditions, the relative CL intensity of iodophenol blue-catalyzed CL system was linearly dependent on the concentration of H2O2 in the range 0.025-10 μM (Fig. 7). The regression equation for the signal I (relative CL intensity) was I=827.08+2979C (H2O2) [μM], with a correlation coefficient 0.997 (n=3). The LOD and LOQ was defined as the concentration of H2O2 which gave a CL intensity 3× and 10× the standard deviation of a blank (which was detected eight times parallelly). LOD (3 σ/s) and LOQ (10 σ/s) values 11
were found to be 0.014 and 0.022 μM, respectively. In addition, a series of 8 repetitive measurements of 5, 1 and 0.25 μM H2O2 were employed for estimating the precision. The relative standard deviation was 2.58%, 5.16% and 4.66% for 5, 1 and 0.25 μM H2O2, respectively. (Figure 7) In order to investigate the selectivity of the proposed CL method, varying amounts of possible interferents were added to solution of 5 μM H2O2 and the CL signal was recorded. The tolerable concentration of each possible interferent was taken as a 5% relative error in the CL signal. The influence of ions to 5 μM H2O2 was first evaluated in the absence of EDTA. The results were listed in Table 1. The most serious interference was observed with Fe ions. EDTA, as a chelate reagent, was then added to sample solution to eliminate the interference derived from Fe ions. The experimental results demonstrated that the influence of Fe ions is highly diminished by the addition of 0.0001 M EDTA. The recovery of Fe ions in the presence of 0.0001 M EDTA was 96.32% indicating that the addition of EDTA to sample solution should be advantageous. (Table 1) 3.5. Assay performance for glucose detection Hydrogen peroxide is the main product of GOx-catalyzed reaction. Therefore, the proposed CL method was applied for CL detection of glucose. The CL detection of glucose can be performed in two steps: 1) GOx catalyzes the reaction between glucose and oxygen resulting in the formation of gluconic acid and H 2O2; 2) the generated H2O2 reacts with iodophenol blue and luminol to produce light.
12
The detailed procedure is described in the experimental section. Fig. 8A shows the linear range of glucose detected in PBS buffer which is from 0.1 to 30 μM with a detection limit of 0.06 μM. The regression equation for the signal I (relative CL intensity) was I=3385+2060C (glucose) [μM], with a correlation coefficient 0.989 (n=3). To evaluate the selectivity of the method, control experiments were performed using buffer solution, D-lactose, D-xylose, and D-mannose. A high CL response was observed when 1 μM of lucose was tested, while negligible signals were obtained using buffer solution, 1 μM of D-lactose, D-xylose, and D-mannose (Fig. 8B). The results show promising selectivity of the proposed CL assay for glucose. Finally, the proposed CL method was used for the determination of glucose in diluted human serum samples (Table 2). A validation of the method was carried out by the glucose paper method as a reference method whose results were also listed in Table 2. As shown in Table 2, there is a good agreement between the proposed CL method and the glucose paper method, indicating the capability of the proposed CL method being applied to the determination of glucose in biological sample with satisfactory results. (Figure 8) (Table 2)
4. Conclusion
In summary, iodophenol blue was found to catalyze the luminol-H2O2 CL reaction in this study. The luminophor of the iodophenol blue catalyzed luminol-H2O2 CL system was 3-APA*. The mechanism study showed that 13
iodophenol blue served as catalyst to accelerate the generation of oxidizing radical species such as OH and O2-, and subsequent formation of 1O2 which reacted with luminol anion to emit light. Based on the novel CL system, a simple, sensitive and selective CL assay for H 2O2 was developed. The proposed method demonstrated high sensitivity that can detect as low as 14 nM H 2O2. The proposed CL assay was applied for the determination of glucose in diluted human serum by combining with glucose oxide. The results of CL detection of glucose were in good agreement with those obtained by a commercial paper-based method. The results demonstrate the potential application of the proposed method in determination of enzyme-coupled catalyzed biologically important molecules, such as uric acid, cholestrerol, and lactic acid. Table 3 compared the metrological parameters and green aspects of the proposed method with reported methods indicating that the proposed method is simple, sensitive, and applicable. This work not only displays a new property of iodophenol blue in CL field and enriches luminol CL mechanism but also shows great potential applications in analytical and biochemical fields.
(Table 3)
Acknowledgment
This study was supported by National Natural Science Foundation of China (21475094) and the 973 project (nos. 2015CB856500). Reference
14
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[30] O. Nozaki, M. Munesue, and H. Kawamoto, Luminescence 22 (2007) 401-406. [31] A. Szterk, I. Stefaniuk, B. Cieniek, M. Kuzma, Nukleonika 58 (2013) 447-450. [32] D. M. Wang, Y. Zhang, L. L. Zheng, X. X. Yang, Y. Wang, and C. Z. Huang, J. Phys. Chem. C. 116 (2012) 21622-21628. [33] M. Y. Zhang, Q. L. Sheng, F. Nie, and J. B. Zheng, J. Electroanal. Chem. 730 (2014) 10-15. [34] A. K. Dutta, S. Das, P. K. Samanta, S. Roy, B. Adhikary, and P. Biswas, Electrochimica. Acta 144 (2014) 282-287. [35] E. Canbay, B. Sahin, M. Kiran, and E. Akyilmaz, Bioelectrochemistry 101 (2015) 126-131. [36] X. W. Zheng, and Z. J. Zhang, Anal. Lett. 32 (1999) 3013-3028. [37] T. Y. Yeh, C. I. Wang, and H. T. Chang, Talanta 115 (2013) 718-723. [38] J. F. Ping, J. Wu, K. Fan, and Y. B. Ying, Food Chem. 126 (2011) 2005-2009. Figure captions Fig. 1 The structure of iodophenol blue. Fig. 2 Kinetic characteristics (A) and CL spectra (B) of the luminol-H2O2 CL system in the absence (a) or presence (b) of iodophenol blue. Experimental conditions for (A): 0.25 mM iodophenol blue in 0.1 M HAc-KAc buffer solution (pH 6.5), 10 μL; 5 μM H2O2, 40 μL; 0.5 mM luminol in 0.1 M Tris buffer (pH 10.0), 50 μL; for (B): 5 mM H2O2, 40 μL; 5 mM luminol, 50 μL; other experimental conditions were the same as (A).
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Fig. 3 ESR spectra of nitroxide radicals generated by the reaction of TEMP probe with 1O2. Conditions: modulation amplitude, 1.00 G; microwave power, 20.000 mW; receiver gain, 5.02e + 003; sweep width, 150 G. The ESR measurements were achieved with a Bruker A300 spectrometer operating in the X-band at room temperature. Fig. 4 ESR spectra of DMPO-OH adduct in the luminal-H2O2 system in the presence or absence of iodophenol blue. Condition: modulation amplitude, 1.00 G; microwave power, 20.020 mW; receiver gain, 5.02e + 005; sweep width, 200.000 G. The ESR measurements were achieved with a Bruker EMX spectrometer operating in the X-band at room temperature. Fig. 5. The Kinetic characteristics of the lumimol-H2O2-iodophenol blue system. Experimental conditions were the same as Fig. 2A. Fig. 6 Effects of the reactant conditions on luminol-H2O2 CL system in the presence of iodophenol blue. (A) Effect of luminol concentration on relative CL intensity. Experimental conditions: iodophenol blue in 0.1 M HAc-KAc buffer (pH 6.5), 10 μL; 5 μM H2O2, 40 μL; different concentrations of luminol in 0.1 M Tris buffer (pH 10), 50 μL. (B) Effect of Tris buffer pH on CL signal. Experimental conditions: 0.5 mM luminol in 0.1 M Tris buffer (PH 8-12), 50 μL; other experimental conditions were the same as Fig. 6A. (C) Effect of HAc-KAc buffer pH on CL signal. Experimental conditions: 0.1 mM iodophenol blue in 0.1 M HAc-KAc buffer solution (PH 5.5-7.5), 10 μL; other experimental conditions were the same as Fig. 6B. (D) Effect of
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iodophenol blue concentration on CL signal. Experimental conditions: different concentrations of iodophenols blue in 0.1 M HAc-KAc buffer (pH 6.5), other experimental conditions were the same as Fig. 6C. Fig. 7 Calibration curve of CL intensity vs concentration of H2O2. The inset shows the CL responses of H2O2 detection at low concentrations. Experimental conditions: 0.25 mM iodophenol blue in 0.1 M HAc-KAc buffer (pH 6.5), 10 μL; different concentrations of H2O2, 40 μL; 0.5 mM luminol in 0.1 M Tris buffer (pH 10), 50 μL. Fig. 8 (A) CL response of the luminol-glucose/GOx-iodophenol blue system in the prensence of different concentrations of glucose. Experimental conditions: luminol, 0.5 mM; iodophenol blue, 0.25 mM; GOx, 10U; glucose, 0.1, 0.5, 1, 10, 20, 30 μM. (B) Selectivity of the sensing for glucose compared to other possible interfering targets. The concentration of glucose, PBS, D-lactose, D-xylose and D-mannose is 10 μM. Other experimental conditions were the same as denoted in Fig. 8 (A). Table 1 Recoveries of 5 μM H2O2 in the presence of interferents Coexisting species
Concentration (mg L-1)
Recovery (%)
Na+
23
101.13
Mn2+
27.5
95.67
K+
19.5
103.47
Fe3+
28
40.37
Ag+
54
98.66
Cl-
36
98.58
NH4+
18
100.66
Ac-
22
103.47
Ca2+
20
99.13
HCO3-
30
97.88
Mg2+
12
99.62
H2PO4-
48
98.67
Co2+
29.5
98.58
NO3-
31
98.66
19
Coexisting Concentration Recovery (mg L-1) species (%)
Cu2+
32
SO42-
98.53
48
100.66
Table 2 Results of determination of glucose in diluted serum Proposed method1)
Glucose paper method
/mmol L-1
/mmol L-1
1
3.01 ± 0.41
2.9
2
5.63 ± 0.46
6.0
3
2.01 ± 0.17
2.3
4
7.51 ± 0.51
8.6
5
10.20 ± 1.63
9.7
Sample
1)
± Expanded uncertainty (U) for k = 2
Table 3 Comparision of sensitivity for different H2O2 assay methods H2O2 detection
Drawbacks
Analytical method
Detection
Application
Linearity
A
B
Ĝ
limit
range
Electrochemistry[29]
32.6 μM
80.0-372.0 mM
-
Ĝ
Electrochemistry [30]
1.1 μM
10.0-1900 μM
H2O2 in human urine
Ĝ
Electrochemistry [31]
10 nM
0.1-70 μM
-
Ĝ
CL [32]
0.35 μM
1.0-4000 μM
-
CL [10]
11 nM
0.1-5.0 μM
-
CL
14 nM
this study
glucose in human serum
20
Ĝ
C
Phosphorescence [33]
140 nM
1.0-60 μM
glucose in blood and saliva
Fluorimetry [22]
0.34 μM
5.0-200 μM
L-lactate in human serum
Amperometry [34]
0.05 μM
0.1-120 μM
H2O2 in beverages
Ĝ
Ĝ
A represents complex modification or synthesis processes are involved. B represents use of toxic organic reagent or toxicity unknown nanoparticles. C represents a time-consuming detection procedure was needed.
Highlights 1. A novel luminol CL system was developed and applied to the detection of hydrogen peroxide. 2. The working range is 0.025-10 μM and the detection limit is 14 nM. 3. The new CL assay exhibits high sensitivity and extraordinary specificity.
4. The proposed method has been applied to the detection of glucose in diluted serum.
21
Ĝ
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
*Graphical Abstract (for review)