A novel biosensor based on DNA hybridization for ultrasensitive detection of NOS terminator gene sequences

Sensors and Actuators B 257 (2018) 538–544

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A novel biosensor based on DNA hybridization for ultrasensitive detection of NOS terminator gene sequences Yanhua He, Zhefeng Fan ∗ Department of Chemistry, Shanxi Normal University, Linfen 041004, PR China

a r t i c l e

i n f o

Article history: Received 18 August 2017 Received in revised form 29 October 2017 Accepted 30 October 2017 Keywords: Biosensor Graphene oxide quantum dots NOS terminator gene sequences DNA hybridization Graphene oxide

a b s t r a c t In this work, we have successfully designed a novel biosensor based on DNA hybridization for ultrasensitive detection of NOS terminator gene sequences (NOSt). This biosensor was synthesized by connecting single-stranded capture DNA (sDNA)-labeled graphene oxide quantum dots (GOQDs) (QDs-sDNA) as fluorescent probe and graphene oxide (GO) as quencher. The detection principle based on hybridization combinations can occur between QDs-sDNA and complementary target DNA; moreover, QDs-sDNA can bind to GO with significantly higher affinity than QDs-dsDNA. In the absence of complementary target DNA, QDs-sDNA was absorbed onto the surface of GO, and the fluorescence of QDs-sDNA was quenched due to fluorescent resonant energy transfer. In the presence of a complementary target DNA, its hybridization with QDs-sDNA formed QDs-dsDNA, which cannot be adsorbed to the GO surface and this leads to reduced quenching. By comparing the fluorescence intensity of QDs-sDNA and QDs-dsDNA in the presence of GO, we can achieve target DNA detection. Thus, rapid, simple, sensitive, efficient, and eco-friendly detection of NOSt was realized. This biosensor had a detection limit of 0.008 nM and a linear range of 0.05–50 nM. Moreover, this sensor can selectivity detect target DNA compared with random and singlebase-mismatched sequences, and was successfully applied to the determination target DNA sequences in biological fluids directly. This sensor can be applied to detect other target DNA sequences by simply changing the types of sDNA coupled to the GOQDs. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Recently, biotechnology advances and increased demand for crop production have led to the rapid development of transgenic technology and popularization of genetically modified organisms (GMOs) [1–3]. However, with the rapid development of the number and diversity of GMOs, safety problems of genetically modified foods, potential ecological pollution problems, and ethical issues have aroused wide spread concern in the community and caused controversy [4,5]. Therefore, sensitive detection of GMOs is important. NOS terminator gene sequences (NOSt), which are derived from the NOS gene of Agrobacterium tumefaciens, are often used as an insert for GMOs and a biomarker of transgenic plants [6,7]. Therefore, sensitive NOSt detection for GMO detection is convenient. In the past few years, various methods have been developed for NOSt detection, such as electrochemical detection [6,8,9], gene chip and GLSS [10], dynamic light scattering (DLS) [11], and realtime PCR [12]; however, these methods still have drawbacks. With

∗ Corresponding author. E-mail address: [email protected] (Z. Fan). https://doi.org/10.1016/j.snb.2017.10.183 0925-4005/© 2017 Elsevier B.V. All rights reserved.

the increased GMO problems, finding a rapid, simple, sensitive, efficient and eco-friendly method for detecting NOSt is urgent. Fluorescent resonant energy transfer (FRET) has been proven to be a sensitive and selective method in biological detection [13–17]. FRET is a near-field energy transfer from a fluorescent donorto a fluorescent acceptor within a close proximity [18,19]. Choosing a pair of good donors and receptors is particularly important for improving the efficiency of FRET. Graphene oxide quantum dots (GOQDs), a novel carbon-based nanomaterial [20], have unique properties, such as easy preparation, easy modification, low toxicity, good biocompatibility, and light stability, and have been widely used in bioimaging [21], light-emitting [22], and environmental fields [23]. However, to the best of our knowledge, GOQDs modified as a fluorescent probe combined with FRET, used in biological detection at a very early stage. GO, is a two-dimensional carbon crystal with one-atom thickness, which is an excellent fluorescent quencher [24–26], has a higher affinity for single-stranded DNAs (sDNAs) than doublestranded DNAs (dsDNAs) [27]. Hence, GO-based fluorescent sensors have been widely used to design sDNA detection based on the mechanism of DNA hybridization [13,28,29]. Using the “postmixing” strategy (let the sDNA probe hybridize with the com-

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plementary target DNA and then add GO) [30,31], given its rapid and sensitive detection, is becoming popular. However, detecting NOSt using this strategy has not been reported. Taking the advantage of the exceptional properties of GOQDs and GO, as well as easy preparation of the single-stranded capture DNA (sDNA)-labeled GOQDs (QDs-sDNA), we report a DNA-based sensor for detecting NOSt, wherein QDs-sDNA acted as the energy donor and recognition probe and GO acted as the receptor. The mechanism of detection was due to hybrid combinations occurring between QDs-sDNA and target DNA and QDs-sDNA binding to GO with significantly higher affinity than QDs-dsDNA. In the absence of target DNA, GO can absorb QDs-sDNA onto its surface, and the fluorescence of QDs-sDNA is quenched. However, in the presence of target DNA and due to the hybridization between QDs-sDNA and target DNA (forming QDs-dsDNA), the FRET between QDs-dsDNA and GO cannot occur and the degree of fluorescence quenching is greatly reduced. Thus, the fluorescence intensities of the QDsdsDNA/GO and QDs-sDNA/GO systems are different and the degree of distinction is proportional to the amount of target DNA added. Following this mechanism, target DNA detection can be achieved. 2. Experimental 2.1. Materialsand chemicals Citric acid (CA), H2 SO4 , NaNO3 , KMnO4 , NaCl, KCl, sodium citrate, and graphite powder were purchased from Guangfu Chemicals Company (Tianjin, China). 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimide ester (NHS) were purchased from GL Biochem Ltd. (Shanghai, China). Ultrafiltration tube (3 KD) and dialysis bags (MW:1000) were supplied by Union Carbide Co. (USA). SSC (1.5 mM sodium citrate, 0.15 mM NaCl, and pH 7.8) was used as a washing and binding buffer. Ultrapure water (18.2 M) from the United States Milli-Q purification system was used throughout the experiment. All oligonucleotides related to NOSt (Table S1) were synthesized and purified by HPLC in Sangon Biotech (Shanghai) and protected with TE buffer (10 mM Tris-HCl, 1 mM EDTA, and pH 8.0) placed at −20 ◦ C until use.

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poured into the beaker, and filter cake was thoroughly washed GO was then washed with distilled water until pH = 5 and then dried. GOQDs were synthesized using an easy “bottom-up” method [32]. Briefly, 2 g CA was placed in a 5 ml beaker and heated to 200 ◦ C. During heating, CA was first liquefied. The liquid color changed from colorless to pale yellow and finally turned orange after 30 min. This color transition implied the formation of GOQDs. The obtained orange liquid was dialyzed in a dialysis bag with a 1000 Da molecular weight cut off against deionized water for 48 h to remove excess CA. Then, the solution in the bag was freeze dried to obtain the solid GOQDs. 2.4. Synthesis of QDs-sDNA The resulting QDs solution (1 mg/ml, 1 ml) was first activated for 2 h in the presence of EDC (20 mM, 1 ml) and NHS (200 mM, 1 ml) at room temperature. Then, 30 ␮g (1.0 OD) sDNA (MW:8212.5) was added and the mixture was further incubated for 24 h at 4 ◦ C. Thus, the amino groups of the sDNA and carboxyl groups of the QDs covalently bonded. The reaction solution was centrifuged with ultrafiltration tube (3KD) at 8000 rpm for 20 min three times to remove uncoupled QDs and other small molecules.The filtrate was diluted with SSC (pH 7.8) to 15 ␮g/ml and kept at 4 ◦ C until use. 2.5. Detection of target DNA QDs-sDNA (15 ␮g/ml, 1 ml) and a series of different concentrations of target DNA (0–50 nM) were added to the colorimetric tubes (10 ml), mixed thoroughly, and hybridized for 30 min at 37 ◦ C. Then, GO (2 mg/ml, 100 ␮l) was added into the above mixtures; the mixtures were diluted with SSC (pH 7.8) to 5 ml, shaken evenly, placed at room temperature, and incubated for 10 min. The fluorescence signals of these solutions were measured by a fluorescent spectrophotometer under an excitation wavelength of 365 nm. Slit widths of excitation measured 5 nm and slit widths of emission reached 10 nm. To prevent background interference, the signal output value was calculated from the relative fluorescence intensity FF0 /F0 ; F0 and F represent the fluorescence intensity when the target DNA is absent and present, respectively.

2.2. Instruments

2.6. Real sample assay

Morphological structures of QDs were characterized using a Tecnai G2 F20 electron microscope (FEI, USA). Fluorescence measurements were performed using aLS-55 luminescence spectrophotometer. Ultraviolet–visible (UV–vis) absorption spectra were performed on a Cary 300 UV–vis Spectrophotometer (Varian, USA). Fourier transform infrared (FT-IR) spectra were obtained on a Nicolet 380 FT-IR spectrometer at 4000–400 cm−1 wavelength range.

The practicality of the sensor was studied by using the tissue extract of plant Glycine max (L.) Merr as the experimental material. DNA was extracted by phenol–chloroform–isoamyl alcohol and washed with ethanol to remove impurities, and the precipitate was dissolved in TE buffer (pH 8.0) and placed at −20 ◦ C until used. The DNA extract was allowed to stand at 95 ◦ C for 10 min, cooled in an ice bath for 5 min, and finally diluted 100 times with SSC (pH 7.8) for detection.

2.3. Synthesis of GO and GOQDs

2.7. Probe stability study

The GO in this study was synthesized according to the modified Hummers method [16]. Briefly, 115 ml concentrated H2 SO4 was added to a beaker and allowed to cool to 0 ◦ C. A total of 5 g graphite powder, 2.5 g NaNO3 , and 15 g KMnO4 were slowly added to the beaker. Temperature was kept at 0 ◦ C, and mixture was stirred continuously. After 2 h, mixture was kept in 35 ◦ C water bath and stirred for 30 min. A total of 230 ml distilled water was then added slowly, and temperature was maintained at temperatures not higher than 98 ◦ C. The beaker was placed in an oil bath at 98 ◦ C for 15 min. Enough warm distilled water was then prepared. The mixture turned brown and was diluted to 700 ml with warm water then poured onto 12.5 ml of 30% H2 O2 . Resulting mixture changed from brown to gold. A total of 5% HCl was hot-filtered and

To study the stability of the QDs-sDNA probe, we recorded the fluorescence intensity of the new probe and divided the solution into two parts. A part of the solution was subjected to the following experiment: QDs-sDNA (15 ␮g/ml, 1 ml) and target DNA (0 nM, 10 nM) were added to the colorimetric tubes (10 ml), mixed thoroughly, and hybridized for 30 min at 37 ◦ C. Then, GO (2 mg/ml, 100 ␮l) was added into the above mixtures; the mixtures were diluted with SSC (pH 7.8) to 5 ml, shaken evenly, placed at room temperature, and incubated for 10 min; moreover, the fluorescence intensity was recorded. The other part of the solution was placed for 2 weeks at 4 ◦ C; the same treatment was performed, and the fluorescence intensity was recorded. Each experiment was repeated thrice.

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Fig. 1. (A)TEM image and (B) particle size distribution of QDs; (C)fluorescence excitation spectra (a) and emission spectra (b) of QDs; inset: the photographs of QDs under visible light (left),under UV light irradiation (right); (D) FT-IR spectra of QDs.

3. Results and discussion 3.1. Characterization of QDs The shape and size of GOQDs were characterized by a transmission electron microscope (TEM), which showed that the particle size distribution of the QDs was uniform and the size was approximately 2.3 nm (Figs. 1A and B). Fig. 1C illustrates the typical excitation and emission of the obtained QDs; the QDs had the largest excitation peak at 365 nm (curve a), and the largest emission peak at 460 nm (curve b). Inset in Fig. 1C shows the photographs of GOQDs dispersion in SSC (pH 7.8) under visible light (left) and UV light irradiation (right). The solution is colorless under visible light and blue in UV light irradiation. In Fig. 1D, absorption values of GOQDs at 3400, 2923, 2410, 1640, and 1388 cm−1 are characteristic of O H, C H stretching vibration, C O stretching vibration, and O H deformation peak. These results indicate that GOQDs contain numerous carboxyl groups that can covalently couple with amino groups of sDNA. 3.2. Spectral characterization of QDs and QDs-sDNA Fig. 2A shows the UV–vis absorption spectra of QDs and QDssDNA. Both have the same absorption peaks at approximately 365 nm. Compared with the absorption spectrum of QDs, a new absorption peaks at approximately 260 nm was found in the absorption spectra of QDs-sDNA; this finding indicated that the QDs had good coupling with DNA [33]. Fig. 2B shows the fluorescence emission spectra of QDs and QDs-sDNA. The fluorescence intensity of the QDs-sDNA is slightly lower than that of QDs. This is because in order to ensured that all sDNAs were coupled with GOQDs in this coupling experiment to avoid excessive sDNA self-folding, thus affecting the experiment sensitivity. We used an excess of QDs, and after the coupling reaction, we conducted centrifugation to remove

uncoupled QDs. however, the emission wavelength range of the two are basically the same (380–630 nm); GO can quench fluorescence of all visible regions [26]; hence, an efficient energy transfer between QDs-sDNA and GO can take place.

3.3. Working principle of DNA-based sensor detection The detection principle is illustrated in Scheme 1. The carboxyl groups of the QDs are covalently bound to the amino groups of the sDNA under the activation of EDC/NHS to form QDs-sDNA and then absorbed onto the surface of GO; this result was due to the aromatic and hydrophobic rings of sDNA that were bound to GO through hydrophobic interactions and ␲–␲ stacking [28,34]; then the distance between QDs-sDNA and GO is narrowed, which led the transfer of QDs-sDNA fluorescence to GO by FRET. In Fig. 3 (comparison between curves a and b), the presence of GO (40 ␮g/ml) induces a remarkable fluorescence quenching efficiency (86.2%) to QDs-sDNA. By contrast,in the presence of target DNA, its selective binding to QDs-sDNA became a highly stable structure (QDsdsDNA) due to the hidden DNA base inside the helical structure that cannot be used for surface bonding for a long time; moreover, only negatively charged phosphate groups were exposed [35]. Furthermore, QDs-dsDNAcannot be adsorbed to the GO surface, FRET cannot occur, and fluorescence quenching significantly decreased. In Fig. 3 (comparison between curves b and c), when 10 nM target DNA was added, the fluorescence intensity of the QDs-dsDNA/GO systemis significantly higher than that of QDs-sDNA/GO system, and the difference in fluorescence intensities is proportional to the amount of target DNA. In this way, we can use this sensor to achieve quantitative detection of target DNA.

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Fig. 2. (A) UV–vis absorption spectrum of QDs and QDs-sDNA; (B) fluorescence emission spectra of QDs and QDs-sDNA.

pled with QDs. Hence, the QDs-sDNA concentration was expressed in the sDNA concentration in this paper. 3.5. The sensor performance for DNA detection

Fig. 3. Fluorescence spectra of (a)QDs-sDNA, (b) QDs-sDNA/GO, and (c) QDsdsDNA/GO.

Under optimum conditions, we recorded the fluorescence emission spectrum change of this system upon adding different concentrations of target DNA (0–50 nM). In Fig. 4A, the fluorescence intensity of the system gradually increased with the increase of target DNA concentration. We established a linear relationship between fluorescence intensity and DNA concentration (Fig. 4B). This biosensor has linear target DNA detection ranges of 0.05–0.3 and 0.3–50 nM with linear equations F − F0 /F0 = 2.578t DNA + 0.956 (R2 =0.995) and F − F0 /F0 = 0.027 tDNA + 1.876 (R2 =0.998), respectively, and a detection limit (LOD) of 0.008 nM. LOD is defined by the equation LOD = 3/s, where  is the standard deviation of the corrected blank signals when no target DNA in the sensor is found, and s represents the slope of calibration curve. 3.6. The sensor selectivity and interference detection

3.4. Optimization of experimental conditions To obtain the best experimental results, we optimized important factors that may affect the experimental results, including QDssDNA amount, GO, temperature, and reaction time. In Fig. S1A, the value of F- F0 /F0 gradually increased with increasing QDs-sDNA and peaked when concentration of QDs-sDNA reached 3 ␮g/ml. However, with further increase in QDs-sDNA, the value of F- F0 /F0 decreased. This is due to the fact that large concentrations of quantum dots can only add to their self-priming phenomenon, resulting in background disturbances [36,37]. Thus, 3 ␮g/ml of QDs-sDNA was selected as the optimal concentration. On this basis, we optimized the concentration of GO. In Fig. S1B, when the concentration of GO is 40 ␮g/ml, the value of F- F0 /F0 is at peak; hence, we chose 40 ␮g/ml GO for follow-up experiments. Fig. S1C shows the effect of reaction temperature on DNA hybridization. At 37 ◦ C, the hybridization effect and biological activity of DNA are at their highest; hence, the hybridizations were fastest [37,38]. Fig. S1D is the result of the optimization involving added GO fluorescence quenching time (Time1) and the DNA hybridization time (Time2). The quenching effect improved with time and peaked at 10 min. With prolonged time, the quenching effect no longer improved. Thus, we chose 10 min as the best quenching time; in the same manner, we selected 30 min as the best DNA hybridization time. It is worth mentioning that, to avoid excessive sDNA bending, which affects the experimental results, excessive QDs were used in coupling reactions. Afterward, the reactions were centrifuged to remove QDs that were not conjugated and the waste liquid showed no absorption peaks at 260 nm by UV–vis. Therefore, we believe that all sDNA were cou-

To evaluate the selectivity of the novel sensing system, we added the same amount (10 nM) of NOSt, MT1 , MT2 , MT3 , and Nt separately into this new probe. The relative fluorescence intensities (FF0 /F0 ) upon adding 10 nM of MT1 , MT2 , MT3 , and Nt were approximately 32.1%, 21.6%, 13.2%, and 4.3% of the relative fluorescence intensity (F- F0 /F0 ) obtained upon adding the same amount of target DNA (Fig. S2A). Such significant differences indicated that Nt (Noncomplementary target DNA), MT1 (Single-base-mismatched target DNA), MT2 (Two-base-mismatched target DNA), and MT3 (Threebase-mismatched target DNA) had lower hybridization abilities toward QDs-sDNA compared with the target DNA. This result is similar with the previous reports [28,39–41]. Therefore this sensor can selectively and effectively detect target DNA. We further carried out the interference study of our fluorescent biosensor to various coexisting substances, which included some metal ions and biomolecules commonly present in biological fluids. In Fig. S2B, with the presence of 10 nM target DNA, the 2000-fold for K+ , Na+ , Ca2+ , Mg2+ , and 5000-fold l-cysteine (L-Cys), l-histidine (L-His), L-glycin (L-Gly), and glucose (Glu) did not significantly affect the sensitivity of this DNA sensor (<5%). These results indicate that the sensitivity of the probe is not substantially affected by other potential substances in biological fluids. 3.7. Detection of target DNA in plant samples Furthermore, we added the target DNA (5, 10, and 30 nM) to the plant tissue extract (G.max (L.) Merr) and recorded the changes in the fluorescence intensity of the probe to verify that this new biosensor can detect the target DNA sequence in plants tissue

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Fig. 4. (A)Fluorescence emission spectra of this biosensor with different concentrations of NOSt; (B) linear relationship of this biosensor with different concentrations of NOSt.

Scheme 1. Sensing mechanism of the proposed biosensor for target DNA.

Table 1 Detection of NOS terminator gene sequences in plant tissue extract samples. Type of samples

Concentration (nM)

Detected Concentration (nM)

Recovery (%) (Mean ± s; n = 3).

Glycine max (L.) Merr tissue extracts

5 10 30

4.87 10.21 31.03

97.3 ± 4.2 102.1 ± 3.5 103.4 ± 4.5

extract. The equation was calibrated to calculate the mean and spiked recovery within 97.3%–103.4%. Results indicated that this DNA-based sensor can be applied to biological fluidsand does not need pre-treatments (Table 1). 3.8. The sensor stability detection We compared the fluorescence intensities of the new probe (QDs-sDNA) with the same probe placed 2 weeks at 4 ◦ C. In Table S2, after 2 weeks, the fluorescence intensity of the probe changed 4.3%, and when GO and target DNA were added, the fluorescence intensity trends were the same with those of the new probes. Comparing the fluorescence intensitiesof the QDs-sDNA/GO mixture of

the new probe with the one placed for 2 weeks at 4 ◦ C, and comparing the fluorescence intensity of the QDs-dsDNA/GO mixture of the new probe with the one placed for 2 weeks at 4 ◦ C, the differences were within the tolerable range (<5%). Results indicated that the probe has good stability; moreover, 2 weeks after being placed at 4 ◦ C, fluorescence intensity slightly changed and the probe can still be used normally. 3.9. Advantages of this DNA-based sensor In Table 2, in comparison with other methods of detecting the NOS terminator, the LOD of our method is similar or superior than those of other methods [6,10], except for the DLS method [11]; how-

Table 2 Comparison of methods applied in the principle of DNA hybridization for DNA detection. Method Electrochemical Gene chip and GLSS Gold NPs and DLS Hairpin probes labeled FAM Fluorescence of CuInS2 RTP of Mn-doped ZnS QDs Fluorescence of the AgNCs Fluorescently labeled hairpins Fluorescence of GOQDs

Detection target NOSt NOSt NOSt Not reported H. pylori Ca MV 35S HIV Viral DNA NOSt

Linear range −12

Detection limit −9

8.0 × 10 –4.0 × 10 M Not reported 1.0 × 10−13 –5.0 × 10−9 M 2–40 nM 1.25–875 pM 12–300 nM 1–50 nM Not reported 0.05–50 nM

−12

2.75 × 10 M 44 nM −14 3.0 × 10 M 0.7 nM 0.46 pM 4.03 nM 0.4 nM 5 nM 0.008 nM

Reference [6] [10] [11] [17] [28] [38] [42] [43] This work

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ever, our method has a wider linear range than DLS. Moreover, our method does not need complex sample pretreatment and is not interfered by light scattering and auto fluorescence. In comparison with other methods from recent publications on detecting different DNAs, our method offers advantages, such as low LOD [17,38,42,43], wide linear range [17,28,42], and most importantly, the most ecofriendly. Hence, the sensor we developed can efficiently, rapidly, and sensitively detect NOSt, in an eco-friendly manner, and can be used for detecting other target DNA sequences by simply changing the types of sDNA coupled to the GOQDs. 4. Conclusion In conclusion, we have successfully used a fluorescent DNA sensor to detect GMO marker gene NOS terminators for the first time. This biosensor enables rapid, sensitive, and eco-friendly detection of target DNA in biological fluids, and does not need complex pretreatment. The high performance of this process can be attributed to the following: one-step synthesis of eco-friendly and carboxylrich GOQDs and high quenching efficiency of GO; simple QDs-sDNA preparation; the QDs-sDNA probe can hybridize with the target DNA and then add GO to significantly shorten the detection time; the probe has good stability, selectivity, specificity, and can avoid the interferences of auto fluorescence and light scattering. In addition to these attributes, this work expands GOQD applications in fluorescent probes.

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Appendix A. Supplementary data [26]

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2017.10.183.

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Biographies Yanhua He is currently carrying out her graduate work for her doctor degree underthe guidance of Professor Fan in Shanxi Normal University. Her research focuses on the synthesis and functionalization of quantum dots and their application on biosensors for biomolecules and small molecules.

Zhefeng Fan is a professor at the Department of Chemistry, Shanxi Normal University. He received his doctor degree from Wuhan University (China) in 2005. His research focuses on application of nanomaterials in spectral and chromatographic analysis.