H2O2-mediated fluorescence quenching of double-stranded DNA templated copper nanoparticles for label-free and sensitive detection of glucose

Article abstract of DOI:10.1039/c5ra14852a

Blood glucose monitoring has attracted extensive attention because diabetes mellitus is a worldwide public health problem. Here, a novel and label-free fluorescent sensing strategy was reported for simple and sensitive detection of glucose in human serum on the basis of H₂O₂-mediated fluorescence quenching of double-stranded DNA (ds-DNA) templated copper nanoparticles (Cu NPs). In this strategy, the fluorescence intensity of ds-DNA templated Cu NPs was found to be quenched effectively in the presence of H₂O₂. Similarly, glucose could be monitored based on the enzymatic conversion of glucose by glucose oxidase to generate H₂O₂. Under the optimized conditions, the strategy exhibited sensitive and selective detection of glucose in a linear range from 50 nM to 100 μM and with a detection limit of 12 nM. In addition, the method was successfully applied in the detection of glucose in human serum samples with satisfactory results. Furthermore, the strategy was free of any fluorescence dye label, complex DNA sequence design, and sophisticated experimental techniques. Therefore, the proposed approach could hold great potential for diabetes mellitus research and clinical diagnosis.

Full text of DOI:10.1039/c5ra14852a

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DOI: 10.1039/C5RA14852A
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H O -mediated fluorescence quenching of double-stranded
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DNA templated copper nanoparticles for label-free and
sensitive detection of glucose
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Hai-Bo Wang*, Hong-Ding Zhang, Ying Chen, Yang Li, Tian Gan
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College of Chemistry and Chemical Engineering, Xinyang Normal University,
Xinyang 464000, PR China
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* Corresponding author. Tel.: +86 376 6391172; Fax: +86 376 6391172.
E-mail address: wanghaibohn@163.com (H.-B. Wang).
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Abstract
Blood glucose monitoring has attracted extensive attention because diabetes
mellitus is a worldwide public health problem. Here, a novel and label-free
fluorescent sensing strategy was reported for simple and sensitive detection of glucose
in human serum on the basis of H O -mediated fluorescence quenching of
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double-stranded DNA (ds-DNA) templated copper nanoparticles (Cu NPs). In this
strategy, the fluorescence intensity of ds-DNA templated Cu NPs was found to be
quenched effectively in the presence of H O . Similarly, glucose could be monitored
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based on the enzymatic conversion of glucose by glucose oxidase to generate H O .
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Under the optimized conditions, the strategy exhibited sensitive and selective
detection of glucose in a linear range from 50 nM to 100 μM and with a detection
limit of 12 nM. In addition, the method was successfully applied in the detection of
glucose in human serum samples with satisfactory results. Furthermore, the strategy
was free of any fluorescence dye label, complex DNA sequence design, and
sophisticated experimental techniques. Therefore, the proposed approach could hold
great potential for diabetes mellitus research and clinical diagnosis.
Keywords: Glucose detection; H O ; Label-free fluorescent sensor; Copper
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nanoparticles
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1. Introduction
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As a major component of animal and plant carbohydrates, glucose plays a key
role in living systems. It acts not only as an important energy source of the living cells,
but also as a metabolic intermediate in the synthesis of other complex molecules. The
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blood glucose levels are also an important indicator of human health conditions. The
deficiency of glucose may result in hypoglycemia. On the other hand, a high level of
glucose is connected to many other diseases such as diabetes, hypertension and
cardiovascular diseases. About 300 million people suffered from diabetes in the year
2010; and this number is estimated to almost double in 2030 by the statistics of the
World Health Organization. Thus, it is of great importance to be able to accurately
monitor the blood glucose levels in clinical diagnosis of diabetes. Several
conventional methods including spectrophotometry, fluorometry, chemiluminescence
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and electrochemistry have been developed for the monitoring blood glucose levels.
Although these methods were quite powerful, they usually suffered from some
disadvantage. The spectrophotometry and electrochemistry-based methods were
limited by the interference of blood color and contamination of the electrode by the
proteins in blood. Among these methods, fluorescent method was used widely due to
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its operational simplicity and high sensitivity. Quantum dots (QDs) become one of
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the most popular fluorescence materials in such application.
However, most of
previous strategies usually suffered from complicated modification or harsh detection
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environment. Furthermore, the cytotoxic effect of the probes could not be neglected.
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Thus, it is still highly desirable to develop simple, low-cost and sensitive
approaches for the determination of glucose.
In contrast to the conventional organic dyes and quantum dots, fluorescent metal
nanoclusters or nanoparticles have attracted significant attention in the field of
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bioanalysis due to their unique electrical, optical properties and low toxicity.
Among the various reported metal nanoclusters or nanoparticles, DNA or
oligonucleotide templated fluorescent copper nanoparticles as a type of newly
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emerged functional biochemical probe, have possessed great potential as fluoDOreI:
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probes for biochemical applications because of their advantages of good
biocompatibility, low-cost, ease of preparation, low-toxicity and excellent
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fluorescence property.
Recently, Mokhir et al. reported that ds-DNA could act as an efficient template
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for the formation of Cu NPs through the reduction of Cu by ascorbic acid and the
formed Cu NPs exhibited excellent fluorescence, whereas ss-DNA template did not
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support the formation of Cu NPs. The formation of ds-DNA templated Cu NPs
contained two steps. The first step in the reaction was the reduction of Cu (II) to Cu (I)
followed by the disproportionation of Cu (I) into Cu (II) and Cu (0). The second step
was that the formed Cu (0) was clustered on ds-DNA scaffolds. Furthermore, ds-DNA
templated Cu NPs could be facilely prepared within 5 minutes at room temperature.
Thus, due to its simplicity, high efficiency, rapidity, and hypotoxicity, the ds-DNA
templated Cu NPs have been used as fluorescence probes in some biological
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assays.
Chen et al. found that Pb could quench the fluorescence of ds-DNA
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Hu et al. have utilized ds-DNA templated Cu NPs as novel fluorescence probe for
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label-free detection of biothiols based on the quenching of their fluorescence. The
quenching effect was ascribed to the coordination complex formed by the Cu-S
metal-ligand bond between the Cu NPs and the biothiols. Our group found that
dopamine could also effectively quench the fluorescence of Cu NPs by the formation
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of photo-induced electron transfer process between dopamine and Cu NPs. However,
exploration of fluorescence quenching of ds-DNA templated Cu NPs is still at a very
early stage.
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Interestingly, we found that the fluorescence intensity of ds-DNA templated Cu
NPs could be quenched effectively by H O in this study. Moreover, glucose could be
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oxidized by dissolved oxygen (O ) in the presence of glucose oxidase (GOD) to
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produce glucose acid and H O . Then, the concentration of glucose could be obtained
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by detecting the amount of the enzymatically generated H O , which quenc
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fluorescence of Cu NPs. The principle of our proposed fluorescent sensor for
hydrogen peroxide (H O ) and glucose detection was schematically represented in
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Scheme 1. The ds-DNA templated Cu NPs were used as fluorescent indicator. In the
absence of hydrogen peroxide, the formed ds-DNA templated Cu NPs exhibited
excellent fluorescence intensity. However, it was found that the fluorescence intensity
of the ds-DNA templated Cu NPs could be quenched effectively by the presence of
H O (shown in Scheme 1A). The fluorescence quenching mechanism was discussed
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in ESI†. Based on the intensive quenching effects, H O could be successfully
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detected through the fluorescence change of ds-DNA templated Cu NPs. In addition,
by taking advantage of H O as a mediator, this strategy was further exploited for
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constructing oxidase-based biosensors for glucose detection. As shown in Scheme 1B,
glucose was oxidized by dissolved oxygen (O ) in the presence of glucose oxidase to
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produce glucose acid and H O . Consequently, the glucose concentration could be
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determined indirectly by the amount of enzymatically generated H O according to
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the fluorescence quenching. Hence, a novel and cost-effective fluorescent sensor was
constructed for sensitive detection of glucose based on the H O -mediated
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fluorescence quenching of ds-DNA templated Cu NPs.
(Scheme 1)
126 2. Experimental
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2.1. Reagents
All oligonucleotides in this work were synthesized by Sangon Biotechnology Co.
Ltd. (Shanghai, China) and used without further purification. The sequences of these
oligonucleotides were shown as follows: P1 5'-CAT AGC GGC AGG ATC AGT TAC
AGT G-3'; P2: 5'-CAC TGT AAC TGA TCC TGC CGC TAT G-3'. Glucose oxidase,
CuSO ·5H O, H O (30%) and ascorbic acid were purchased from Sigma-Aldrich
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All the water used in this work was obtained from a Millipore Milli-Q water
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2.2. Apparatus
The fluorescence measurements were performed on a Hitachi F-7000
fluorescence spectrometer (Hitachi Co. Ltd., Japan) equipped with a Xenon lamp
excitation source. A quartz fluorescence cell with an optical path length of 10 mm was
used. The excitation wavelength was set at 340 nm, and the fluorescence emission
spectra of Cu NPs were collected from 500 nm to 640 nm at room temperature with
both the excitation and emission slit set at 5.0 nm. All fluorescence measurements
were carried out at room temperature unless stated otherwise.
2.3. Synthesis of ds-DNA templated Cu NPs
The ds-DNA templated Cu NPs were synthesized according to the literature with
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a slight modification.
Briefly, a mixture solution containing 1 μM P1 and 1 μM
P2 ss-DNA in MOPS buffer (20 mM MOPS, 300 mM NaCl, pH 7.0) was firstly
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prepared. Then the mixture was denatured at 95 C for 10 min, and subsequently
cooled down slowly to room temperature to ensure that P1 and P2 DNA were
completely hybridized to form ds-DNA. After that, 10 μL 2.5 mM CuSO solutions
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and 10 μL 30 mM ascorbic acid solutions were added into the mixture solution and
kept for 5 minutes at room temperature to form ds-DNA templated Cu NPs. Finally,
the fluorescent spectrum of the mixture was recorded by F-7000 spectrophotometer
(Hitachi Co. Ltd., Japan) immediately. The morphology of ds-DNA templated Cu NPs
was characterized by transmission electron microscope (TEM) and shown in Fig. 1.
And the size of ds-DNA templated Cu NPs was about 3-5 nm.
(Fig. 1)
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2.4. Fluorescence quenching effect by H O2
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In a typical measurement, different concentrations of H O were freshly prepared
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before use. Then, 10 μL different concentrations of H O were added into 90 μL
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as-prepared ds-DNA Cu NPs, and the mixture was incubated at room temperature for
10 min in the dark. After that, the fluorescence intensity of the mixture was
immediately measured by F-7000 spectrophotometer with the excitation wavelength
of 340 nm.
2.5. Label-free detection of glucose
In a typical assay of glucose, a 10 μL mixture solution (20 mM MOPS, 300 mM
NaCl, pH 7.0) containing 0.05 mg/mL glucose oxidase and different concentrations of
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glucose were incubated at 37 C for 30 min. After that, the above mixture was added
into 90 μL prepared Cu NPs and incubated at room temperature for 10 min in the dark.
At last, the fluorescence intensities of the reaction solution were measured by F-7000
spectrophotometer with the excitation wavelength of 340 nm.
183 3. Results and discussion
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3.1. Evaluation the quenching effect of H O2
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In order to evaluate the feasibility of the strategy, the fluorescence intensity of
obtained Cu NPs were tested in the absence and presence of H O . As shown in Fig. 2,
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it was observed that ds-DNA templated Cu NPs exhibited excellent fluorescence at
565 nm in the absence of H O (curve a in Fig. 2). However, the fluorescence
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intensity decreased significantly (curve b in Fig. 2) after the addition of H O . These
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results indicated that H O could strongly quench the fluorescence intensity of
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ds-DNA templated Cu NPs. Based on the quenching effect, a simple and label-free
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fluorescent assay could be developed for H O detection by using the d
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templated Cu NPs.
(Fig. 2)
It has been reported that the fluorescence intensity of ds-DNA templated Cu NPs
was obviously pH dependent and relatively low in acidic solutions. Thus, the effect of
pH value on the fluorescence response was studied. The efficiency of fluorescence
quenching was calculated by F /F, where F and F were the fluorescence intensity of
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ds-DNA templated Cu NPs in the absence and presence of 10 μM H O , respectively.
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As shown in Fig. 3A, the fluorescent quenching efficiency increased gradually in the
pH value range from 4.0 to 7.0 and then decreased when the pH value was higher than
7.0. A remarkable response was obtained at pH 7.0. Therefore, pH 7.0 was suitable
for this sensing system.
Furthermore, the incubated time of H O was another important parameter
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influencing the fluorescent intensity. So, the effect of incubated time of H O was also
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investigated to improve the sensitivity of this strategy. As shown in Fig. 3B, the
fluorescence intensity decreased obviously in the presence of 10 μM H O . And there
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was no obvious change in the fluorescence intensity of ds-DNA templated Cu NPs
after 10 min. Thus, the incubated time of H O was set at 10 min.
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To further demonstrate the fluorescence quenching ability of H O , the
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fluorescence intensity of ds-DNA templated Cu NPs at different concentrations of
H O was investigated under the optimized experimental condition. The fluorescence
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spectra of the ds-DNA templated Cu NPs in the presence of variable concentrations of
H O were shown in Fig. 4A. It was found that the fluorescence intensity of ds-DNA
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templated Cu NPs decreased with the H O concentration increasing from 10 nM to
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50 μM. Fig. 4B depicted the relationship between the H O concentration and the
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fluorescence intensity at the maximum emission wavelength. As shown in inset of Fig.
4B, a good linear relationship was obtained in the concentration range from 10 nM to
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nM. The detection limit was comparable to or better than those of previously reported
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fluorescence methods.
(Fig. 4)
3.2. Fluorescent detection of glucose
The fluorescence quenching of Cu NPs by H O enabled the implementation of
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the ds-DNA templated Cu NPs as versatile fluorescence indicators for sensitive
detection of the activity of O -dependent oxidases and their substrates. For example,
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glucose could be oxidized by oxygen (O ) in the presence of glucose oxidase (GOx)
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to generate H O . Thus, it is also possible for the detection of glucose by
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H O -mediated fluorescence quenching of ds-DNA templated Cu NPs.
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Since glucose and glucose oxidase were all essential to produce H O , some
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parameters (such as concentration of glucose oxidase and incubated time of glucose
oxidase) were optimized to achieve the sensitive detection of glucose. Firstly, the
effect of glucose on the fluorescence intensity was investigated. It was found that no
significant change of the fluorescence intensity were observed when Cu NPs only
mixed with glucose, indicating that glucose had little effect on the fluorescence
intensity of Cu NPs. Secondly, the effect of the concentration of glucose oxidase on
the fluorescence quenching was also studied. The efficiency of fluorescence
quenching was calculated by F /F, where F and F were the fluorescence intensity of
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ds-DNA templated Cu NPs in the absence and presence of glucose, respectively. As
shown in Fig. 5A, the efficiency of fluorescence quenching reached a maximum value
when the glucose oxidase concentration was 0.05 mg/mL. Thus, 0.05 mg/mL glucose
oxidase was selected as the optimized concentration. Additionally, the incubated time
of glucose oxidase was another important parameter influencing the fluorescent
intensity. So, the effect of incubated time of glucose oxidase was investigated to
improve the sensitivity of this strategy. It could be seen from Fig. 5B that the
fluorescence intensity increased rapidly and then approached a plateau after 30 min.
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Therefore, 30 min of the glucose oxidase incubated time was used through
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(Fig. 5)
Fig. 6 depicted the typical fluorescent assay of glucose on the basis of
H O -mediated fluorescence quenching of ds-DNA templated Cu NPs. It could be
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seen from Fig. 6A, the fluorescent intensity decreased gradually with increasing
concentrations of glucose range from 50 nM to 500 μM, which suggested that the
higher the glucose concentration being added, the more H O was generated. Fig. 6B
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illustrated the relationship between the glucose concentration and the fluorescence
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intensity. According to fluorescence quenching model of Stern-Volmer,
the F /F
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range from 50 nM to 100 μM (shown in the inset of Fig. 6B). Where, F and F were
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the fluorescence intensity of sensing system in the absence and presence of glucose,
respectively. The detection limit was calculated to be 12 nM based on three times the
standard deviation rule (3σ), which was comparable to or better than that of most
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previously reported methods.
Thus, these results demonstrated that the proposed
method could be applied to sensitively determinate glucose.
(Fig. 6)
3.3. Selectivity of glucose
In order to demonstrate the selectivity of the present strategy toward glucose,
other possible interfering substances were investigated, such as various saccharides,
amino acids, ascorbic acid (AA), and uric acid (UA). As shown in Fig. 7, except for
glutathione (GSH) and cysteine (Cys), these substances did not result in obvious
interference in glucose detection. This was because the thiol group of GSH and Cys
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could also quench the fluorescence intensity of Cu NPs.
Thus, a masking agent
(N-ethylmaleimide, NEM) was introduced into the sensor system to eliminate
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interference from GSH and Cys. After incubation of GSH or Cys with NEM, a
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negligible fluorescence response was observed, whereas glucose detecti
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unaffected by the introduction of NEM. The results suggested that the proposed assay
exhibited high selectivity and could be used for determination of glucose in biological
samples.
(Fig. 7)
3.4. Detection of glucose in human serum samples
To verify the feasibility of our new approach for detection of glucose in
biological samples, we applied it to analyze glucose in healthy human blood serum
samples provided by Xinyang Central Hospital (Xinyang, China). Taking into
consideration the normal glucose level in healthy human blood as well as the linear
range of our method, the blood serum samples were diluted 20 times. Then, 0.3 mM
NEM was added into the samples to eliminate the interference from GSH and Cys in
real samples. The results were presented in Table 1. The glucose concentrations of the
serum samples were coincided with those provided by local hospital. In order to
determine the accuracy and precision of the method, appropriate amounts of glucose
standards were added to the human serum sample (shown in Table S1 of ESI†). The
results revealed that proposed method was feasible for practical blood glucose
monitoring in real samples.
(Table 1)
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In conclusion, we have developed a label-free and sensitive fluorescent biosensor
for glucose detection on the basis of H O -mediated fluorescence quenching of
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ds-DNA templated Cu NPs. Due to the excellent quenching ability of H O , the
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label-free sensor exhibited sensitive and selective detection of glucose with a
detection limit of 12 nM. The method was also applied to monitor glucose levels in
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human serum with satisfactory results, suggesting that our approach ha
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potential for diabetes mellitus research and clinical diagnosis. The strategy was
convenient and without complicated preparation procedure. Furthermore, the
proposed strategy provided an alternative platform to detect other substrates through
oxidation by its O -dependent oxidase which could generate H O . Thus, it could
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offer a new approach to developing low-cost and sensitive methods for biological and
clinical diagnostics applications.
321 Acknowledgements
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This work was financially supported by National Natural Science Foundation of
China (No. 21305119), Foundation of Henan Educational Committee (No.
13A150768).
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Figure captions
Scheme 1 (A) Schematic illustration of fluorescence quenching of ds-DNA templated
Cu NPs by H O . (B) Schematic illustration of fluorescent strategy for glucose
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detection based on H O -mediated fluorescence quenching of ds-DNA templated Cu
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NPs.
Fig. 1 TEM image of ds-DNA templated Cu NPs.
Fig. 2 Fluorescence spectra of ds-DNA templated Cu NPs in the absence (curve a) and
presence of 10 μM H O (curve b), respectively.
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Fig. 3 (A) The effect of pH values on the fluorescence responses of the sensing
system. Where F and F were the fluorescence intensity of ds-DNA templated Cu NPs
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in the absence and presence of 10 μM H O , respectively. (B) The effect of H O
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incubation time on the fluorescence intensity.
Fig. 4 (A) Fluorescent spectra of ds-DNA templated Cu NPs in the presence of
different concentrations of H O , the curves from top to bottom, the concentration of
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H O were 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 μM, respectively. (B) The
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relationship between the fluorescence intensity and H O concentration. Inset of (B) is
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the calibration curve. The error bars represent the standard deviation of three
measurements.
Fig. 5 (A) The effect of glucose oxidase on the fluorescence response. Where, F and
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F were the fluorescence intensity of sensing system in the absence and presence of
glucose, respectively. (B) The effect of glucose oxidase incubation time on the
fluorescence intensity.
Fig. 6 (A) Fluorescent spectra of sensing system in the presence of different
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Fig. 7 The selectivity of H O -mediated fluorescence quenching for glucose assay.
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Where, F and F are the fluorescence intensity of sensing system in the absence and
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presence of glucose and other analytes, respectively. The error bars represent the
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Table captions
Table 1 Determination of glucose levels in the human serum samples (n = 3).
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Table 1
Samples
Proposed method (mM)
RSD (%)
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Local hospital (mM)
Relative deviation (%)
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10 nm
Fig. 1