Biosensors and Bioelectronics 54 (2014) 372–377
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel HBV genotypes detecting system combined with microfluidic chip, loop-mediated isothermal amplification and GMR sensors Xiao Zhi a, Min Deng a, Hao Yang b, Guo Gao a, Kan Wang a, Hualin Fu a, Yixia Zhang a, Di Chen a, Daxiang Cui a,n a
National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People’s Republic of China b Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai, Beijing 100071, P.R. China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 September 2013 Received in revised form 28 October 2013 Accepted 6 November 2013 Available online 15 November 2013
Genotyping of hepatitis B virus (HBV) can be used for clinical effective therapeutic drug-selection. A novel microfluidic biochip for HBV genotyping has been fabricated, for the first time, integrating loopmediated isothermal amplification (LAMP), line probes assay (LiPA) and giant magnetoresistive (GMR) sensors. Coupling LAMP with LiPA in microfluidic chip shortened reaction time substantially, and combining LAMP with GMR sensor enabled limit of detection to attain 10 copies mL 1 target HBV DNA molecules in 1 h. Furthermore, the independent designed GMR sensors and microfluidic chip can decrease manufacturing cost and patient's test-cost, and facilitate GMR detector repeating use for signal detection. In addition, the detection system has a lower background signal owing to application of superparamagnetic nanoclusters. And it can be expected to use for multiple target molecules synchronous detection in microfluidic chip based on a characteristic of stationary reaction temperature of LAMP. In conclusion, the neoteric detecting system is well suitable for quick genotyping diagnosis of clinical HBV and other homothetic biomolecule detection in biological and medical fields. & 2013 Elsevier B.V. All rights reserved.
Keywords: HBV genotypes Microfluidic chip LAMP GMR sensors Superparamagnetic nanoclusters
1. Introduction Hepatitis B virus (HBV), discovered in 1966, is a major cause of chronic hepatitis, hepatocirrhosis and hepatocellular carcinoma (HCC) (Lee, 1997). HBV infection is a highly endemic disease in China. There are an approximate 93 million HBV carriers, and among them 20 million are patients with chronic hepatitis B (Hepatology, 2011; Lu and Zhuang, 2009) Hepatitis B virus (HBV) can be classified into ten genotypes (A–J) based on intergenotype divergence of 8% or more in the entire nucleotide sequence (Hussain et al., 2003; Olinger et al., 2008; Tatematsu et al., 2009; Tran et al., 2008). Statistics have shown that genotype A, B, C and D are found in China, and genotype B (41%) and C (53%) are dominant (Zeng et al., 2005). HBV genotypes may influence hepatitis B e antigen (HBeAg) seroconversion rate, mutational pattern in the basal core promoter (BCP) and precore (PC) regions and the course of liver disease (Liu and Kao, 2013; Yang et al., 2008; Yang et al., 2013). Genotyping may be helpful for clinical diagnosis and effective therapeutic drug selection (Biswas et al., 2013; Bonino et al., 2007).
n
Corresponding author. Tel.: þ 86 21 34206886. E-mail address: [email protected] (D. Cui).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.025
Many different genotyping methods have been reported including direct sequencing (Valsamakis, 2007), restriction fragment polymorphism (RELP) (Chen et al., 2013), line probe assay (LiPA) (Guirgis et al., 2010; Qutub et al., 2006), type-specific PCR (Chen et al., 2007; Naito et al., 2001), real-time PCR (Yeh et al., 2004), oligonucleotide microarray chip (Pazienza et al., 2013; Song et al., 2006) and enzymelinked immunosorbent assay for genotype-specific epitopes (Moriya et al., 2002). However, the mentioned-above methods have various problems such as time-consumption, high cost, trivial procedure, low sensitivity, inferior accuracy and specificity. How to develop a perfect technology with low cost, less-time consumption, high sensitivity and specificity for genotyping is still a concern. Loop-mediated isothermal amplification (LAMP) is a well-developed method for rapid amplification of nucleic acid. Because it has high sensitivity based on a strand displacement active enzyme and good specificity depending on specially designed primers recognizing six to eight regions of the target DNA sequence, LAMP has been used to detect pathogenic microorganisms (Ihira et al., 2007; Poole et al., 2012; Qiao et al., 2013; Song et al., 2012), food safety (Duarte et al., 2013; Qu et al., 2013; Wang et al., 2013), genetically modified products (Guan et al., 2010; Li et al., 2013; Zhai et al., 2012), cancer diagnosis (Horibe et al., 2007; Sagara et al., 2013; Wang et al., 2012) and embryo sex identification (Hirayama et al., 2013; Hirayama et al., 2004; Zoheir and Allam, 2010) Giant magnetoresistance (GMR) is a quantum mechanical
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magnetoresistance effect, which was observed in thin-film structures composed of alternating ferromagnetic and non-magnetic conductive layers. The 2007 Nobel Prize in Physics was awarded to Albert Fert and Peter Grunberg for the discovery of GMR (Baibich et al., 1988; Binasch et al., 1989). Up to date, the GMR multilayer film as sensor has been used in different configurations for successful biosensing. For example, measurement of forces at the level of single biomolecule (Baselt et al., 1998; Graham et al., 2003), identifying biological warfare agents (Edelstein et al., 2000), detecting DNA hybridization and pathogenic microorganism (Marquina et al., 2012; Osterfeld et al., 2008; Pazienza et al., 2013), screening diagnostic biomarkers (Bao et al., 2013; Kim et al. 2013a, 2013b), monitoring chemical reaction (Podesva and Foret, 2013), and so on. In our laboratory, we also successfully developed a microfluidic chip system combined with GMR sensors, magnetic nanoclusters (MNCs) and line probe assay (LiPA) for HBV genotyping (Zhi et al., 2012). Nevertheless, the system does not have the capability of high efficient nucleic acid amplification, and the microfluidic chip system integrated GMR sensors, which increase the detection cost and fabrication complexity. In order to overcome these shortcomings and increase chip performance, we ingeniously assemble a neoteric HBV detecting system composed of an independent GMR detector and a microfluidic chip integrated with LAMP and LiPA, and investigated the feasibility of the use of prepared detecting system for rapid HBV genotyping.
2. Materials and methods 2.1. LAMP primers and genotype-specific probes design Conserved LAMP primers were designed by PrimerExplorer V4 program at the website (http://primerexplorer.jp/e/), and genotype specific hybridization probes were designed based on reported references (Cai et al., 2011; Grandjacques et al., 2000; Stuyver et al., 2000; Tomita et al., 2008) and analysis of data of HBV genomic sequences from National Center for Biotechnology Information (NCBI) nucleotide sequence database (GenBank). These primers and probes were provided by Invitrogen life technologies (USA). The primer B3, F3, BIP (B1c B2) and biotinylated primer FIP (F1c–F2) were used to amplify conserved domain of HBV genome. Probe T can hybrid with all different genotypes of HBV DNA, probe B and C respectively uniquely hybrid with genotype B or genotype C of HBV DNA. B3: 5′-cggggtttttcttgttgac-3′ F3: 5′-tgaggcatagcagcaggat-3′ BIP: 5′-tccccctagaaaattgagagaagtaatcctcacaataccacagag-3′ FIP: 5′-Bio-tggccaaaattcgcagtcccaaaacgccgcagacacatc-3′ Probe T: 5′-NH2-(T)15-atcgctggatgtgtctgcggcgtttt-3′ Probe B: 5′-NH2-(T)15-cccaaatctccagtcactcaccaacctgttgt-3′ Probe C: 5′-NH2-(T)15-ggagcacccacgtgtcctggccaaaatt-3′ The target samples for hybridization were LAMP products amplified from clone plasmid HBV DNA with complete genome. These samples approximate real clinical medical samples, and therefore give a direct test case to demonstrate if the detecting system is suitable for clinical applications. HBV plasmids for HBV genotype B and C were kindly provided by Prof. Y.M. Wen (Fudan University, Shanghai, China). 2.2. Probes immobilization and preparation of MNCs Three hybridized probes (14 μM) were immobilized on the clean pretreated slide surface according to related reports (Razumovitch et al., 2009; Zhi et al., 2012). The slide was used for followed fabrication of microfluidic chip.
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Magnetic nanoclusters (MNCs) with 172 nm in diameter were synthesized according to our previous report (Gao et al., 2011; Hu et al., 2010; Hu et al., 2011). The as-prepared MNCs were characterized by SEM, HR-TEM, FTIR, XRD as well as VSM. Surface of prepared MNCs existed carboxyl group. Carboxyl group on the surface of MNCs was directly conjugated with streptavidin, resulting in the formation of streptavidin-modified MNCs for subsequent detecting step. 2.3. Fabrication of GMR detector and microfluidic chip GMR sensors were fabricated according to our previous reports (Feng et al., 2011; Zhi et al., 2012). GMR multilayers were deposited by dc magnetron sputtering onto 3 in.-Si wafers with the structure of NiFeCo 6 nm[Cu 2.1 nm/NiFeCo 1.5 nm] 10/Ta 100 nm. The sensors based on the GMR multilayers were fabricated by lithography, ion beam etching and lift-off technology. There are four GMR sensors for detecting including three different probe groups and one blank control group (NTC). The resistance of each sensor is 3 kΩ. The detection area of each sensor with the feature line width of 5 μm is 100 μm 100 μm in zigzag pattern (Fig. 1(A-a)). The sensors were covered with a 200 nm thick sputtered SiO2 passivation layer to protect it from environmental corrosion. And then GMR sensors were welded on a printed circuit board (PCB) for easy connection with a digital multimeter (Fig. 1A). The microfluidic chip was fabricated in the good biocompatible polydimethylsiloxane (PDMS) by soft lithography (Chen et al., 2011a; Liu et al., 2010). Firstly, a 1 mm thickness pure glass was chosen as the substrate, and a Cr/Cu seed layer with 100 nm in thickness was sputtered on the glass. Subsequently, the glass was coated by spinning a layer of positive photoresist about 100 μm in thickness, and then was exposed and developed. Afterwards nickel with about 100 μm in thickness was electroformed and photoresist was removed. Then, PDMS pre-polymer was poured over the mother mold and baked at 70 1C for 3 h. Lastly, PDMS was released from the mother mold and punched through to function as inlets/outlet for sample injection and extraction, and PDMS was bonded with a glass slide after oxygen plasma treatment. In this way, microchannel in the microfluidic-chip with 300 μm in length, 300 μm in width and 100 μm in depth was formed successfully (Chen et al., 2011b; Yang et al., 2010). 2.4. LAMP amplification and nucleic acid hybridization LAMP reactions contained 1.6 μM each FIP and BIP, 0.2 μM each F3 and B3, 1.4 μM each dNTP, 1 M Betaine (Sigma), 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Triton X-100, 8U Bst DNA polymerase large fragment (New England Biolabs) and 1 μL HBV DNA template. The mixture was incubated at 63 1C for 1 h. In the meantime, oligonucleotide probes immobilized on the bottom of microchannel hybridized with target HBV DNA fragments produced by LAMP. The schematic of experiment is shown in Fig. 2. After that, streptavidin conjugated magnetic nanoclusters solution (5 μg μL 1) was injected into microchannel, and then, the streptavidin on the surface of magnetic nanoclusters combined with biotin at the terminal of target DNA after incubation at room temperature for 15 min, and then washed the microchannel for 3 min. 2.5. Magnetic signal detection by GMR detector Firstly, the GMR detector was placed in an external vertical magnetic field of 230 Oe, and PCB was connected with a digital multimeter (Agilent 34401A, USA) under 0.1 mA direct current to measure resistance values of GMR sensors (Fig. 1). When no microfluidic chip was settled on the sensors of GMR detector, the average resistance value of GMR sensors was regarded as reference value of detecting system. After LAMP amplification,
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Fig. 1. Structure of the entire detecting system: (A) GMR detector: (a) SEM pattern of GMR. (a) GMR detector: (a) SEM pattern of GMR sensor; (b) microfluidic-chip: (b, c) SEM images of negative region and positive region after LAMP and hybridization; (c) Signal output instrument.
resistance values of GMR sensors were measured respectively before and after injecting streptavidin conjugated magnetic nanoclusters solution into microfluidic chip. The values of twice the measurements were regarded as detection value. If there is a significant change between detection value and reference value (P o0.05), which means target HBV DNA were detected by the detecting system and refers to positive signal. If there is no significant change between detection value and reference value (P 40.05), which means there were no target HBV DNA were detected by the detecting system and refers to negative signal. 2.6. Statistical analysis All results are reported as means 7SD. All statistical tests were done with SPSS 21.0 (IBM Corp. USA). Comparison for two groups was performed using student t test. The level of P o0.05 was regarded as significant.
for fabricating disposable microfluidic chip was modified with genotype-specific probes by a chemical method (Supplementary data, Scheme 1). 3.1. Characterization of MNCs We successfully prepared magnetic nanoculsters (MNCs) with a nearly spherical shape and uniform size of c.a. 172 nm (Supplementary data, Fig. S1A, B, C and Fig. S2), and that showed typical X-ray diffraction (XRD) patterns of magnetite (Supplementary data, Fig. S1D). The surface of MNCs was coated with carboxyl group (Supplementary data, Fig. S3), and the MNCs had magnetization saturation values of 43 em μg 1 at 300 K (Supplementary data, Fig. S4). The prepared magnetic nanoclusters were very stable in PBS solution, no aggregation was observed, After MNCs reacted with streptavidin (Supplementary data), streptavidins were conjugated on the surface of MNCs successfully. 3.2. Analysis of LAMP products
3. Results and discussion As shown in Fig. 1, the novel HBV genotype detecting system was successfully assembled, which consists of disposable microfluidic chip, GMR detector and signal output instrument. And the slide
The mixture of LAMP reagents and DNA template were injected into the microchannel, LAMP and probes-capturing courses occurred simultaneously in the same microchannel (Fig. 2) at 63 1C for 1 h (Asiello and Baeumner, 2011; Mori et al., 2013; Mori
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Fig. 2. Graphic of LAMP and principle of genotyping in microchannel in disposable microfluidic chip.
and Notomi, 2009). As shown in Fig. 3, LAMP products of B and C genotype plasmids gave a similar classic ladder-like pattern in 0.7% agarose gel electrophoresis analysis (Notomi et al., 2000). 3.3. Analysis of the performance of HBV genotypes detecting system In order to test the performance of HBV genotyping detecting system, firstly genotype B and C's HBV plasmids as target DNA for LAMP were prepared. In the course of LAMP, different genotype probes captured matched target DNA molecules, then, magnetic nanoclusters (MNCs) were bound on the products of hybridization through the combined reaction between streptavidin and biotin as shown in Fig. 2, the concrete detection results were shown in Table 1. When T and B regions have positive signals, meanwhile NTC and C regions have negative signals, which means that the detected sample is genotype B HBV DNA (Fig. 4(a)). When T and C regions have positive signals, meanwhile NTC and B regions have negative signals, which means that the detected sample is genotype C HBV DNA (Fig. 4(b)). When T, B and C regions have positive signals, meanwhile NTC region has a negative signal, which means that the sample detected contain genotypes B and C HBV DNAs. When T region has a positive signal, meanwhile NTC, B and C regions have negative signals, which means that the sample detected contain other genotypes HBV DNA excluded genotypes B and C. When all of NTC, T, B and C regions have positive signals, which means the sample detected has no HBV DNA. When NTC region has a positive signal, no matter what signals of T, B and C regions are, which means that the detection is invalid and the sample need to be detected again. Our results fully show that B and C genotype of HBV can be clearly distinguished by the HBV detecting system. It is well known that 2000 IU mL 1 (104 copies mL 1) of serum HBV DNA titer has been used as an important standard to classify
Fig. 3. Electrophoresis pattern of LAMP product: (M) DNA Marker; (1) control; (2) LAMP product of B genotype plasmid; (3) LAMP product of C genotype plasmid.
Table 1 Resistance changes of sensors for B and C genotype samples (n¼ 4, X 7 S.D.) Genotype samples
Signal change value, in mΩ (positive signals in bold) NTC region T region
Genotype B Genotype C n
B region n
C region nn
1.25 7 8.17 131.25±11.54 131.257 8.16 0.25 7 5.77 136.25±10.82n 2.25 7 5.00
11.25 78.16 133.75710.62nn
P o 0.01, NTC region vs. T region. Po 0.01, B region vs. C region.
nn
different stages of chronic hepatitis B in the clinical stage (Hepatology 2011; Keeffe et al., 2008; Lok and McMahon, 2007), therefore, the detection sensitivity and range in 1 h were evaluated by detecting ten-fold serial dilutions (109 copies mL 1– 1 copy mL 1) of genotype C plasmids of HBV. In order to obtain objective information, we collected detection date of HBV plasmid DNA by probe T, based on the experimental result that there were no statistical differences between T region and B region or T region and C region in Fig. 4 (P 40.05). Our results as shown in Fig. 5, HBV DNA from 10 copies mL 1–109 copies mL 1 could be detected, and different genotypes of HBV DNA can be distinguished by using the HBV detecting system. The limit of detection of this established system is 10 copies mL 1 target HBV DNA. Furthermore, there is
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Fig. 4. (a) Detecting for genotype B HBV DNA; (b) Detecting for genotype B HBV DNA.
Fig. 5. Detection sensitivity and range of HBV genotypes detecting.
obvious positive linear relationship between signal intensity and concentration of HBV DNA. In the past several years, a number of new detection methods for HBV genotyping have been reported and used. In 2006, a microfluidic chip coupled with isotachophoresis preconcentration (ITP) and zone electrophoresis (ZE) was used for HBV genotyping. This genotyping method with ITP-ZE chip analysis showed the capability to detect target HBV DNA ranging from 103 to 1011 copies mL 1 (Liu et al., 2006). Zhang et al. (2010) developed a microfluidic device with microbead arrays and DNA probes labeled with quantum dots for genotyping of HBV DNA. The detection sensitivity was 4 pM (2.4 109 copies mL 1). Cai et al. (2011) develpoed a genotype-specific loop-mediated isothermal amplification technique for hepatitis B virus B and C genotyping and quantification. They used two sets of LAMP primers for genotypes B and C. The detection sensitivity of the method were 323 and 515 copies mL 1 for genotypes B and C, respectively . Compared with those methods mentioned-above, our method has obvious advantages such as higher detection sensitivity (10 copies mL 1), greater detection range (10–109 copies mL 1), and only one set of LAMP primers for hepatitis B virus B and C genotyping.
4. Conclusions In conclusion, we successfully established a rapid, accurate, novel HBV genotyping detecting system combined with microfluidic chip, LAMP, LiPA and GMR sensors, which owns marked advantages as follows: (a) Microfluidic chip without integrating
heating and cooling units for nucleic acid amplification obviously decreases fabricating cost and complexity, and future clinical testcost. (b) Owing to that GMR sensors are not integrated into microfluidic chip, the GMR detector can be repeatedly used, the test cost can be further decreased. (c) High sensitivity and specificity of LAMP enhance the performance of the detecting system. With the aid of stationary reaction temperature of LAMP, the microfluidic chip detection system can be used easily for multiple target molecules' synchronous detection in future. (d) Two procedures of amplification and hybridization were finished simultaneously in 1 h, which markedly decreases test time compared with 16 h of single hybridization procedure of gene chip detection. (e) Using superparamagnetic nanoclusters makes the detection exhibit a lower background signal. In short, this low-cost and excellent detecting system is not only suitable for clinical detection, especially in underdeveloped countries, but also lays a foundation for multiple target molecules synchronous detection in the near future.
Acknowledgments This work was supported by the National Major Scientific Projects for the Prevention and Control of HIV/AIDS and Viral Hepatitis of China (No. 2009ZX10004-311), Chinese 973 Project (Grant no. 2010CB933901), the Natural Science Foundation of China (Nos.81225010, 31100717, and 81327002), 863 Project of China (2012AA022703), and Shanghai Science and Technology Fund (Nos.13NM1401500 and 11nm0504200 ).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.025. References Asiello, P.J., Baeumner, A.J., 2011. Lab. Chip 11, 1420–1430. Baibich, M.N., Broto, J.M., Fert, A., Van Dau, F.N., Petroff, F., Etienne, P., Creuzet, G., Friederich, A., Chazelas, J., 1988. Phys. Rev. Lett. 61, 2472–2475. Bao, C.C., Chen, L., Wang, T., Lei, C., Tian, F.R., Cui, D.X., Zhou, Y., 2013. Nano-Micro Lett. 5, 213–222. Baselt, D.R., Lee, G.U., Natesan, M., Metzger, S.W., Sheehan, P.E., Colton, R.J., 1998. Biosens. Bioelectron. 13, 731–739. Binasch, G., Grünberg, P., Saurenbach, F., Zinn, W., 1989. Phys. Rev. B 39, 4828–4830. Biswas, A., Panigrahi, R., Pal, M., Chakraborty, S., Bhattacharya, P., Chakrabarti, S., Chakravarty, R., 2013. J. Med. Virol. 85, 1340–1347. Bonino, F., Marcellin, P., Lau, G.K.K., Hadziyannis, S., Jin, R., Piratvisuth, T., Germanidis, G., Yurdaydin, C., Diago, M., Gurel, S., 2007. Gut 56, 699–705. Cai, Z.J., Lou, G.Q., Cai, T., Yang, J., Wu, N.P., 2011. J. Clin. Virol. 52, 288–294. Chen, J., Chen, D., Yuan, T., Chen, X., 2011a. Nano Biomed. Eng. 3, 203–210.
X. Zhi et al. / Biosensors and Bioelectronics 54 (2014) 372–377
Chen, J.S., Yin, J.H., Tan, X.J., Zhang, H.Q., Zhang, H.W., Chen, B.C., Chang, W.J., Schaefer, S., Cao, G.W., 2007. J. Clin. Virol. 38, 238–243. Chen, L., Bao, C.C., Yang, H., Li, D., Lei, C., Wang, T., Hu, H.Y., He, M., Zhou, Y., Cui, D.X., 2011b. Biosen. Bioelectron. 26, 3246–3253. Chen, Y.M., Wu, S.H., Qiu, C.N., Yu, D.J., Wang, X.J., 2013. Braz. J. Med. Biol. Res. 46, 614–622. Duarte, C., Salm, E., Dorvel, B., Reddy, B., Bashir, R., 2013. Biomed. Microdevices 15, 821–830. Edelstein, R.L., Tamanaha, C.R., Sheehan, P.E., Miller, M.M., Baselt, D.R., Whitman, L.J., Colton, R.J., 2000. Biosen. Bioelectron. 14, 805–813. Feng, J., Wang, Y.Q., Li, F.Q., Shi, H.P., Chen, X., 2011. Detection of magnetic microbeads and ferrofluid with giant magnetoresistance sensors. In: Du, Y.W., Judy, J., Qian, Z., Wang, J. (Eds.), 1st International Symposium on Spintronic Devices and Commercialization. Gao, G., Huang, P., Zhang, Y.X., Wang, K., Qin, W., Cui, D.X., 2011. CrystEngComm 13, 1782–1785. Graham, D.L., Ferreira, H.A., Freitas, P.P., Cabral, J.M.S., 2003. Biosens. Bioelectron. 18, 483–488. Grandjacques, C., Pradat, P., Stuyver, L., Chevallier, M., Chevallier, P., Pichoud, C., Maisonnas, M., Trepo, C., Zoulim, F., 2000. J. Hepatol. 33, 430–439. Guan, X., Guo, J., Shen, P., Yang, L., Zhang, D., 2010. Food Anal. Methods 3, 313–320. Guirgis, B.S.S., Abbas, R.O., Azzazy, H.M.E., 2010. Int. J. Infect. Dis. 14, E941–E953. Hepatology, C.S.o, 2011. Chin. J. Epidemiol. 32, 405–415. Hirayama, H., Kageyama, S., Moriyasu, S., Sawai, K., Minamihashi, A., 2013. J. Reprod. Dev. 59, 321–326. Hirayama, H., Kageyama, S., Moriyasu, S., Sawai, K., Onoe, S., Takahashi, Y., Katagiri, S., Toen, K., Watanabe, K., Notomi, T., 2004. Theriogenology 62, 887–896. Horibe, D., Ochiai, T., Shimada, H., Tomonaga, T., Nomura, F., Gun, M., Tanizawa, T., Hayashi, H., 2007. Int. J. Cancer 120, 1063–1069. Hu, H.Y., Yang, H., Huang, P., Cui, D.X., Peng, Y.Q., Zhang, J.C., Lu, F.Y., Lian, J., Shi, D.L., 2010. Chem. Commun. 46, 3866–3868. Hu, H.Y., Yang, H., Li, D., Wang, K., Ruan, J., Zhang, X.Q., Chen, J., Bao, C.C., Ji, J.J., Shi, D.L., 2011. Analyst 136, 679–683. Hussain, M., Chu, C.J., Sablon, E., Lok, A.S.F., 2003. J. Clin. Microbiol. 41, 3699–3705. Ihira, M., Akimoto, S., Miyake, F., Fujita, A., Sugata, K., Suga, S., Ohashi, M., Nishimura, N., Ozaki, T., Asano, Y., 2007. J. Clin. Virol. 39, 22–26. Keeffe, E.B., Dieterich, D.T., Han, S.H.B., Jacobson, I.M., Martin, P., Schiff, E.R., Tobias, H., 2008. Clin. Gastroenterol. Hepatol. 6, 1315–1341. Kim, D., Lee, J.R., Shen, E., Wang, S.X., 2013a. Biomed. Microdevices. 15, 665–671. Kim, D., Marchetti, F., Chen, Z.X., Zaric, S., Wilson, R.J., Hall, D.A., Gaster, R.S., Lee, J.R., Wang, J.Y., Osterfeld, S.J., 2013b. Sci. Rep., 3. Lee, W.M., 1997. N. Engl. J. Med. 337, 1733–1745. Li, Q.C., Fang, J.H., Liu, X., Xi, X., Li, M.J., Gong, Y.F., Zhang, M.Z., 2013. Eur. Food Res. Technol. 236, 589–598. Liu, B., Deng, Y., Qin, B., Li, Z., He, N., 2010. Nano Biomed. Eng. 2, 149–154. Liu, C.J., Kao, J.H., 2013. Semin. Liver. Dis. 33, 97–102. Liu, D.Y., Shi, M., Huang, H.Q., Long, Z.C., Zhou, X.M., Qin, J.H., Lin, B.C., 2006. J. Chromatogr. B—Anal. Technol. Biomed. Life Sci. 844, 32–38. Lok, A.S.F., McMahon, B.J., 2007. Hepatology 45, 507–539. Lu, F.M., Zhuang, H., 2009. Chin. Med. J. 122, 3–4. Marquina, C., de Teresa, J.M., Serrate, D., Marzo, J., Cardoso, F.A., Saurel, D., Cardoso, S., Freitas, P.P., Ibarra, M.R, 2012. J. Magn. Magn. Mater. 324, 3495–3498. Mori, Y., Kanda, H., Notomi, T., 2013. J. Infect. Chemother. 19, 404–411. Mori, Y., Notomi, T., 2009. J. Infect. Chemother. 15, 62–69.
377
Moriya, T., Kuramoto, I.K., Yoshizawa, H., Holland, P.V., 2002. J. Clin. Microbiol. 40, 877–880. Naito, H., Hayashi, S., Abe, K., 2001. J. Clin. Microbiol. 39, 362–364. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T, 2000. Nucleic Acids Res. 28, e63. Olinger, C.M., Jutavijittum, P., Hubschen, J.M., Yousukh, A., Samountry, B., Thammavong, T., Toriyama, K., Muller, C.P., 2008. Emerg. Infect. Dis. 14, 1777–1780. Osterfeld, S.J., Yu, H., Gaster, R.S., Caramuta, S., Xu, L., Han, S.-J., Hall, D.A., Wilson, R. J., Sun, S., White, R.L., 2008. Proc. Natl. Acad. Sci. 105, 20637–20640. Pazienza, V., Niro, G.A., Fontana, R., Vinciguerra, M., Andriulli, A., 2013. Clin. Chem. Lab. Med. 51, 1707–1717. Podesva, P., Foret, F., 2013. Curr. Anal. Chem. 9, 642–652. Poole, C.B., Tanner, N.A., Zhang, Y.H., Evans, T.C., Carlow, C.K.S, 2012. PLoS. Negl. Trop. Dis. 6, e1948. Qiao, J., Wang, J.W., Meng, Q.L., Wang, G.C., Liu, Y.C., He, Z.H., Yang, H.B., Zhang, Z.C., Cai, X.P., Chen, C.F., 2013. Virol. J. 10, 228. Qu, D.F., Zhou, H.Y., Han, J.Z., Tao, S.Y., Zheng, B.L., Chi, N., Su, C.L., Du, A.F., 2013. Vet. Parasitol. 192, 98–103. Qutub, M.O., Germer, J.J., Rebers, S.P.H., Mandrekar, J.N., Beld, M., Yao, J.D.C., 2006. J. Clin. Virol. 37, 218–221. Razumovitch, J., França, K.d., Kehl, F., Wiki, M., Meier, W., Vebert, C., 2009. J. Phys. Chem. B 113, 8383–8390. Sagara, Y., Ohi, Y., Matsukata, A., Yotsumoto, D., Baba, S., Tamada, S., Sagara, Y., Matsuyama, Y., Ando, M., Rai, Y., 2013. Breast Cancer 20, 181–186. Song, Q.Q., Wang, L.X., Fang, R., Khan, M.K., Zhou, Y.Q., Zhao, J.L., 2012. Trop. Anim. Health Prod. 45, 247–250. Song, Y.J., Dai, E.H., Wang, J., Liu, H.J., Zhai, J.H., Chen, C.Y., Du, Z.M., Guo, Z.B., Yang, R.F., 2006. Mol. Cell. Probes 20, 121–127. Stuyver, L., Van Geyt, C., De Gendt, S., Van Reybroeck, G., Zoulim, F., Leroux-Roels, G., Rossau, R., 2000. J. Clin. Microbiol. 38, 702–707. Tatematsu, K., Tanaka, Y., Kurbanov, F., Sugauchi, F., Mano, S., Maeshiro, T., Nakayoshi, T., Wakuta, M., Miyakawa, Y., Mizokami, M., 2009. J. Virol. 83, 10538–10547. Tomita, N., Mori, Y., Kanda, H., Notomi, T., 2008. Nat. Protoc. 3, 877–882. Tran, T.T., Trinh, T.N., Abe, K., 2008. J. Virol. 82, 5657–5663. Valsamakis, A., 2007. Clin. Microbiol. Rev. 20, 426–439. Wang, L., Shi, L., Su, J.Y., Ye, Y.X., Zhong, Q.P., 2013. Gene 515, 421–425. Wang, T.Z., Zhang, Y., Huang, G.L., Wang, C., Xie, L., Ma, L., Li, Z.Y., Luo, X.B., Tian, H., Li, Q., 2012. Sci. Chin.—Chem. 55, 508–514. Yang, H., Chen, L., Lei, C., Zhang, J., Li, D., Zhou, Z.M., Bao, C.C., Hu, H.Y., Chen, X., Cui, F., 2010. Appl. Phys. Lett. 97, 0437021–0437023. Yang, H.I., Yeh, S.H., Chen, P.J., Iloeje, U.H., Jen, C.L., Su, J., Wang, L.Y., Lu, S.N., You, S.L., Chen, D.S., 2008. J. Natl. Cancer Inst. 100, 1134–1143. Yang, J.H., Zhang, H., Chen, X.B., Chen, G., Wang, X., 2013. World J. Gastroenterol. 19, 3861–3865. Yeh, S.H., Tsai, C.Y., Kao, J.H., Liu, C.J., Kuo, T.J., Lin, M.W., Huang, W.L., Lu, S.F., Jih, J., Chen, D.S., 2004. J. Hepatol. 41, 659–666. Zeng, G., Wang, Z., Wen, S., Jiang, J., Wang, L., Cheng, J., Tan, D., Xiao, F., Ma, S., Li, W., 2005. J. Viral. Hepat. 12, 609–617. Zhai, S.L., Liu, C.X., Zhang, Q.D., Tao, C.Y., Liu, B., 2012. Transgenic Res. 21, 1367–1373. Zhang, H., Xu, T., Li, C.W., Yang, M.S., 2010. Biosens. Bioelectron. 25, 2402–2407. Zhi, X., Liu, Q.S., Zhang, X., Zhang, Y.X., Feng, J., Cui, D.X., 2012. Lab. Chip 12, 741–745. Zoheir, K.M.A., Allam, A.A., 2010. Anim. Reprod. Sci. 119, 92–96.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel HBV genotypes detecting system combined with microfluidic chip, loop-mediated isothermal amplification and GMR sensors Xiao Zhi a, Min Deng a, Hao Yang b, Guo Gao a, Kan Wang a, Hualin Fu a, Yixia Zhang a, Di Chen a, Daxiang Cui a,n a
National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People’s Republic of China b Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai, Beijing 100071, P.R. China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 September 2013 Received in revised form 28 October 2013 Accepted 6 November 2013 Available online 15 November 2013
Genotyping of hepatitis B virus (HBV) can be used for clinical effective therapeutic drug-selection. A novel microfluidic biochip for HBV genotyping has been fabricated, for the first time, integrating loopmediated isothermal amplification (LAMP), line probes assay (LiPA) and giant magnetoresistive (GMR) sensors. Coupling LAMP with LiPA in microfluidic chip shortened reaction time substantially, and combining LAMP with GMR sensor enabled limit of detection to attain 10 copies mL 1 target HBV DNA molecules in 1 h. Furthermore, the independent designed GMR sensors and microfluidic chip can decrease manufacturing cost and patient's test-cost, and facilitate GMR detector repeating use for signal detection. In addition, the detection system has a lower background signal owing to application of superparamagnetic nanoclusters. And it can be expected to use for multiple target molecules synchronous detection in microfluidic chip based on a characteristic of stationary reaction temperature of LAMP. In conclusion, the neoteric detecting system is well suitable for quick genotyping diagnosis of clinical HBV and other homothetic biomolecule detection in biological and medical fields. & 2013 Elsevier B.V. All rights reserved.
Keywords: HBV genotypes Microfluidic chip LAMP GMR sensors Superparamagnetic nanoclusters
1. Introduction Hepatitis B virus (HBV), discovered in 1966, is a major cause of chronic hepatitis, hepatocirrhosis and hepatocellular carcinoma (HCC) (Lee, 1997). HBV infection is a highly endemic disease in China. There are an approximate 93 million HBV carriers, and among them 20 million are patients with chronic hepatitis B (Hepatology, 2011; Lu and Zhuang, 2009) Hepatitis B virus (HBV) can be classified into ten genotypes (A–J) based on intergenotype divergence of 8% or more in the entire nucleotide sequence (Hussain et al., 2003; Olinger et al., 2008; Tatematsu et al., 2009; Tran et al., 2008). Statistics have shown that genotype A, B, C and D are found in China, and genotype B (41%) and C (53%) are dominant (Zeng et al., 2005). HBV genotypes may influence hepatitis B e antigen (HBeAg) seroconversion rate, mutational pattern in the basal core promoter (BCP) and precore (PC) regions and the course of liver disease (Liu and Kao, 2013; Yang et al., 2008; Yang et al., 2013). Genotyping may be helpful for clinical diagnosis and effective therapeutic drug selection (Biswas et al., 2013; Bonino et al., 2007).
n
Corresponding author. Tel.: þ 86 21 34206886. E-mail address: [email protected] (D. Cui).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.025
Many different genotyping methods have been reported including direct sequencing (Valsamakis, 2007), restriction fragment polymorphism (RELP) (Chen et al., 2013), line probe assay (LiPA) (Guirgis et al., 2010; Qutub et al., 2006), type-specific PCR (Chen et al., 2007; Naito et al., 2001), real-time PCR (Yeh et al., 2004), oligonucleotide microarray chip (Pazienza et al., 2013; Song et al., 2006) and enzymelinked immunosorbent assay for genotype-specific epitopes (Moriya et al., 2002). However, the mentioned-above methods have various problems such as time-consumption, high cost, trivial procedure, low sensitivity, inferior accuracy and specificity. How to develop a perfect technology with low cost, less-time consumption, high sensitivity and specificity for genotyping is still a concern. Loop-mediated isothermal amplification (LAMP) is a well-developed method for rapid amplification of nucleic acid. Because it has high sensitivity based on a strand displacement active enzyme and good specificity depending on specially designed primers recognizing six to eight regions of the target DNA sequence, LAMP has been used to detect pathogenic microorganisms (Ihira et al., 2007; Poole et al., 2012; Qiao et al., 2013; Song et al., 2012), food safety (Duarte et al., 2013; Qu et al., 2013; Wang et al., 2013), genetically modified products (Guan et al., 2010; Li et al., 2013; Zhai et al., 2012), cancer diagnosis (Horibe et al., 2007; Sagara et al., 2013; Wang et al., 2012) and embryo sex identification (Hirayama et al., 2013; Hirayama et al., 2004; Zoheir and Allam, 2010) Giant magnetoresistance (GMR) is a quantum mechanical
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magnetoresistance effect, which was observed in thin-film structures composed of alternating ferromagnetic and non-magnetic conductive layers. The 2007 Nobel Prize in Physics was awarded to Albert Fert and Peter Grunberg for the discovery of GMR (Baibich et al., 1988; Binasch et al., 1989). Up to date, the GMR multilayer film as sensor has been used in different configurations for successful biosensing. For example, measurement of forces at the level of single biomolecule (Baselt et al., 1998; Graham et al., 2003), identifying biological warfare agents (Edelstein et al., 2000), detecting DNA hybridization and pathogenic microorganism (Marquina et al., 2012; Osterfeld et al., 2008; Pazienza et al., 2013), screening diagnostic biomarkers (Bao et al., 2013; Kim et al. 2013a, 2013b), monitoring chemical reaction (Podesva and Foret, 2013), and so on. In our laboratory, we also successfully developed a microfluidic chip system combined with GMR sensors, magnetic nanoclusters (MNCs) and line probe assay (LiPA) for HBV genotyping (Zhi et al., 2012). Nevertheless, the system does not have the capability of high efficient nucleic acid amplification, and the microfluidic chip system integrated GMR sensors, which increase the detection cost and fabrication complexity. In order to overcome these shortcomings and increase chip performance, we ingeniously assemble a neoteric HBV detecting system composed of an independent GMR detector and a microfluidic chip integrated with LAMP and LiPA, and investigated the feasibility of the use of prepared detecting system for rapid HBV genotyping.
2. Materials and methods 2.1. LAMP primers and genotype-specific probes design Conserved LAMP primers were designed by PrimerExplorer V4 program at the website (http://primerexplorer.jp/e/), and genotype specific hybridization probes were designed based on reported references (Cai et al., 2011; Grandjacques et al., 2000; Stuyver et al., 2000; Tomita et al., 2008) and analysis of data of HBV genomic sequences from National Center for Biotechnology Information (NCBI) nucleotide sequence database (GenBank). These primers and probes were provided by Invitrogen life technologies (USA). The primer B3, F3, BIP (B1c B2) and biotinylated primer FIP (F1c–F2) were used to amplify conserved domain of HBV genome. Probe T can hybrid with all different genotypes of HBV DNA, probe B and C respectively uniquely hybrid with genotype B or genotype C of HBV DNA. B3: 5′-cggggtttttcttgttgac-3′ F3: 5′-tgaggcatagcagcaggat-3′ BIP: 5′-tccccctagaaaattgagagaagtaatcctcacaataccacagag-3′ FIP: 5′-Bio-tggccaaaattcgcagtcccaaaacgccgcagacacatc-3′ Probe T: 5′-NH2-(T)15-atcgctggatgtgtctgcggcgtttt-3′ Probe B: 5′-NH2-(T)15-cccaaatctccagtcactcaccaacctgttgt-3′ Probe C: 5′-NH2-(T)15-ggagcacccacgtgtcctggccaaaatt-3′ The target samples for hybridization were LAMP products amplified from clone plasmid HBV DNA with complete genome. These samples approximate real clinical medical samples, and therefore give a direct test case to demonstrate if the detecting system is suitable for clinical applications. HBV plasmids for HBV genotype B and C were kindly provided by Prof. Y.M. Wen (Fudan University, Shanghai, China). 2.2. Probes immobilization and preparation of MNCs Three hybridized probes (14 μM) were immobilized on the clean pretreated slide surface according to related reports (Razumovitch et al., 2009; Zhi et al., 2012). The slide was used for followed fabrication of microfluidic chip.
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Magnetic nanoclusters (MNCs) with 172 nm in diameter were synthesized according to our previous report (Gao et al., 2011; Hu et al., 2010; Hu et al., 2011). The as-prepared MNCs were characterized by SEM, HR-TEM, FTIR, XRD as well as VSM. Surface of prepared MNCs existed carboxyl group. Carboxyl group on the surface of MNCs was directly conjugated with streptavidin, resulting in the formation of streptavidin-modified MNCs for subsequent detecting step. 2.3. Fabrication of GMR detector and microfluidic chip GMR sensors were fabricated according to our previous reports (Feng et al., 2011; Zhi et al., 2012). GMR multilayers were deposited by dc magnetron sputtering onto 3 in.-Si wafers with the structure of NiFeCo 6 nm[Cu 2.1 nm/NiFeCo 1.5 nm] 10/Ta 100 nm. The sensors based on the GMR multilayers were fabricated by lithography, ion beam etching and lift-off technology. There are four GMR sensors for detecting including three different probe groups and one blank control group (NTC). The resistance of each sensor is 3 kΩ. The detection area of each sensor with the feature line width of 5 μm is 100 μm 100 μm in zigzag pattern (Fig. 1(A-a)). The sensors were covered with a 200 nm thick sputtered SiO2 passivation layer to protect it from environmental corrosion. And then GMR sensors were welded on a printed circuit board (PCB) for easy connection with a digital multimeter (Fig. 1A). The microfluidic chip was fabricated in the good biocompatible polydimethylsiloxane (PDMS) by soft lithography (Chen et al., 2011a; Liu et al., 2010). Firstly, a 1 mm thickness pure glass was chosen as the substrate, and a Cr/Cu seed layer with 100 nm in thickness was sputtered on the glass. Subsequently, the glass was coated by spinning a layer of positive photoresist about 100 μm in thickness, and then was exposed and developed. Afterwards nickel with about 100 μm in thickness was electroformed and photoresist was removed. Then, PDMS pre-polymer was poured over the mother mold and baked at 70 1C for 3 h. Lastly, PDMS was released from the mother mold and punched through to function as inlets/outlet for sample injection and extraction, and PDMS was bonded with a glass slide after oxygen plasma treatment. In this way, microchannel in the microfluidic-chip with 300 μm in length, 300 μm in width and 100 μm in depth was formed successfully (Chen et al., 2011b; Yang et al., 2010). 2.4. LAMP amplification and nucleic acid hybridization LAMP reactions contained 1.6 μM each FIP and BIP, 0.2 μM each F3 and B3, 1.4 μM each dNTP, 1 M Betaine (Sigma), 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Triton X-100, 8U Bst DNA polymerase large fragment (New England Biolabs) and 1 μL HBV DNA template. The mixture was incubated at 63 1C for 1 h. In the meantime, oligonucleotide probes immobilized on the bottom of microchannel hybridized with target HBV DNA fragments produced by LAMP. The schematic of experiment is shown in Fig. 2. After that, streptavidin conjugated magnetic nanoclusters solution (5 μg μL 1) was injected into microchannel, and then, the streptavidin on the surface of magnetic nanoclusters combined with biotin at the terminal of target DNA after incubation at room temperature for 15 min, and then washed the microchannel for 3 min. 2.5. Magnetic signal detection by GMR detector Firstly, the GMR detector was placed in an external vertical magnetic field of 230 Oe, and PCB was connected with a digital multimeter (Agilent 34401A, USA) under 0.1 mA direct current to measure resistance values of GMR sensors (Fig. 1). When no microfluidic chip was settled on the sensors of GMR detector, the average resistance value of GMR sensors was regarded as reference value of detecting system. After LAMP amplification,
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Fig. 1. Structure of the entire detecting system: (A) GMR detector: (a) SEM pattern of GMR. (a) GMR detector: (a) SEM pattern of GMR sensor; (b) microfluidic-chip: (b, c) SEM images of negative region and positive region after LAMP and hybridization; (c) Signal output instrument.
resistance values of GMR sensors were measured respectively before and after injecting streptavidin conjugated magnetic nanoclusters solution into microfluidic chip. The values of twice the measurements were regarded as detection value. If there is a significant change between detection value and reference value (P o0.05), which means target HBV DNA were detected by the detecting system and refers to positive signal. If there is no significant change between detection value and reference value (P 40.05), which means there were no target HBV DNA were detected by the detecting system and refers to negative signal. 2.6. Statistical analysis All results are reported as means 7SD. All statistical tests were done with SPSS 21.0 (IBM Corp. USA). Comparison for two groups was performed using student t test. The level of P o0.05 was regarded as significant.
for fabricating disposable microfluidic chip was modified with genotype-specific probes by a chemical method (Supplementary data, Scheme 1). 3.1. Characterization of MNCs We successfully prepared magnetic nanoculsters (MNCs) with a nearly spherical shape and uniform size of c.a. 172 nm (Supplementary data, Fig. S1A, B, C and Fig. S2), and that showed typical X-ray diffraction (XRD) patterns of magnetite (Supplementary data, Fig. S1D). The surface of MNCs was coated with carboxyl group (Supplementary data, Fig. S3), and the MNCs had magnetization saturation values of 43 em μg 1 at 300 K (Supplementary data, Fig. S4). The prepared magnetic nanoclusters were very stable in PBS solution, no aggregation was observed, After MNCs reacted with streptavidin (Supplementary data), streptavidins were conjugated on the surface of MNCs successfully. 3.2. Analysis of LAMP products
3. Results and discussion As shown in Fig. 1, the novel HBV genotype detecting system was successfully assembled, which consists of disposable microfluidic chip, GMR detector and signal output instrument. And the slide
The mixture of LAMP reagents and DNA template were injected into the microchannel, LAMP and probes-capturing courses occurred simultaneously in the same microchannel (Fig. 2) at 63 1C for 1 h (Asiello and Baeumner, 2011; Mori et al., 2013; Mori
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Fig. 2. Graphic of LAMP and principle of genotyping in microchannel in disposable microfluidic chip.
and Notomi, 2009). As shown in Fig. 3, LAMP products of B and C genotype plasmids gave a similar classic ladder-like pattern in 0.7% agarose gel electrophoresis analysis (Notomi et al., 2000). 3.3. Analysis of the performance of HBV genotypes detecting system In order to test the performance of HBV genotyping detecting system, firstly genotype B and C's HBV plasmids as target DNA for LAMP were prepared. In the course of LAMP, different genotype probes captured matched target DNA molecules, then, magnetic nanoclusters (MNCs) were bound on the products of hybridization through the combined reaction between streptavidin and biotin as shown in Fig. 2, the concrete detection results were shown in Table 1. When T and B regions have positive signals, meanwhile NTC and C regions have negative signals, which means that the detected sample is genotype B HBV DNA (Fig. 4(a)). When T and C regions have positive signals, meanwhile NTC and B regions have negative signals, which means that the detected sample is genotype C HBV DNA (Fig. 4(b)). When T, B and C regions have positive signals, meanwhile NTC region has a negative signal, which means that the sample detected contain genotypes B and C HBV DNAs. When T region has a positive signal, meanwhile NTC, B and C regions have negative signals, which means that the sample detected contain other genotypes HBV DNA excluded genotypes B and C. When all of NTC, T, B and C regions have positive signals, which means the sample detected has no HBV DNA. When NTC region has a positive signal, no matter what signals of T, B and C regions are, which means that the detection is invalid and the sample need to be detected again. Our results fully show that B and C genotype of HBV can be clearly distinguished by the HBV detecting system. It is well known that 2000 IU mL 1 (104 copies mL 1) of serum HBV DNA titer has been used as an important standard to classify
Fig. 3. Electrophoresis pattern of LAMP product: (M) DNA Marker; (1) control; (2) LAMP product of B genotype plasmid; (3) LAMP product of C genotype plasmid.
Table 1 Resistance changes of sensors for B and C genotype samples (n¼ 4, X 7 S.D.) Genotype samples
Signal change value, in mΩ (positive signals in bold) NTC region T region
Genotype B Genotype C n
B region n
C region nn
1.25 7 8.17 131.25±11.54 131.257 8.16 0.25 7 5.77 136.25±10.82n 2.25 7 5.00
11.25 78.16 133.75710.62nn
P o 0.01, NTC region vs. T region. Po 0.01, B region vs. C region.
nn
different stages of chronic hepatitis B in the clinical stage (Hepatology 2011; Keeffe et al., 2008; Lok and McMahon, 2007), therefore, the detection sensitivity and range in 1 h were evaluated by detecting ten-fold serial dilutions (109 copies mL 1– 1 copy mL 1) of genotype C plasmids of HBV. In order to obtain objective information, we collected detection date of HBV plasmid DNA by probe T, based on the experimental result that there were no statistical differences between T region and B region or T region and C region in Fig. 4 (P 40.05). Our results as shown in Fig. 5, HBV DNA from 10 copies mL 1–109 copies mL 1 could be detected, and different genotypes of HBV DNA can be distinguished by using the HBV detecting system. The limit of detection of this established system is 10 copies mL 1 target HBV DNA. Furthermore, there is
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Fig. 4. (a) Detecting for genotype B HBV DNA; (b) Detecting for genotype B HBV DNA.
Fig. 5. Detection sensitivity and range of HBV genotypes detecting.
obvious positive linear relationship between signal intensity and concentration of HBV DNA. In the past several years, a number of new detection methods for HBV genotyping have been reported and used. In 2006, a microfluidic chip coupled with isotachophoresis preconcentration (ITP) and zone electrophoresis (ZE) was used for HBV genotyping. This genotyping method with ITP-ZE chip analysis showed the capability to detect target HBV DNA ranging from 103 to 1011 copies mL 1 (Liu et al., 2006). Zhang et al. (2010) developed a microfluidic device with microbead arrays and DNA probes labeled with quantum dots for genotyping of HBV DNA. The detection sensitivity was 4 pM (2.4 109 copies mL 1). Cai et al. (2011) develpoed a genotype-specific loop-mediated isothermal amplification technique for hepatitis B virus B and C genotyping and quantification. They used two sets of LAMP primers for genotypes B and C. The detection sensitivity of the method were 323 and 515 copies mL 1 for genotypes B and C, respectively . Compared with those methods mentioned-above, our method has obvious advantages such as higher detection sensitivity (10 copies mL 1), greater detection range (10–109 copies mL 1), and only one set of LAMP primers for hepatitis B virus B and C genotyping.
4. Conclusions In conclusion, we successfully established a rapid, accurate, novel HBV genotyping detecting system combined with microfluidic chip, LAMP, LiPA and GMR sensors, which owns marked advantages as follows: (a) Microfluidic chip without integrating
heating and cooling units for nucleic acid amplification obviously decreases fabricating cost and complexity, and future clinical testcost. (b) Owing to that GMR sensors are not integrated into microfluidic chip, the GMR detector can be repeatedly used, the test cost can be further decreased. (c) High sensitivity and specificity of LAMP enhance the performance of the detecting system. With the aid of stationary reaction temperature of LAMP, the microfluidic chip detection system can be used easily for multiple target molecules' synchronous detection in future. (d) Two procedures of amplification and hybridization were finished simultaneously in 1 h, which markedly decreases test time compared with 16 h of single hybridization procedure of gene chip detection. (e) Using superparamagnetic nanoclusters makes the detection exhibit a lower background signal. In short, this low-cost and excellent detecting system is not only suitable for clinical detection, especially in underdeveloped countries, but also lays a foundation for multiple target molecules synchronous detection in the near future.
Acknowledgments This work was supported by the National Major Scientific Projects for the Prevention and Control of HIV/AIDS and Viral Hepatitis of China (No. 2009ZX10004-311), Chinese 973 Project (Grant no. 2010CB933901), the Natural Science Foundation of China (Nos.81225010, 31100717, and 81327002), 863 Project of China (2012AA022703), and Shanghai Science and Technology Fund (Nos.13NM1401500 and 11nm0504200 ).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.025. References Asiello, P.J., Baeumner, A.J., 2011. Lab. Chip 11, 1420–1430. Baibich, M.N., Broto, J.M., Fert, A., Van Dau, F.N., Petroff, F., Etienne, P., Creuzet, G., Friederich, A., Chazelas, J., 1988. Phys. Rev. Lett. 61, 2472–2475. Bao, C.C., Chen, L., Wang, T., Lei, C., Tian, F.R., Cui, D.X., Zhou, Y., 2013. Nano-Micro Lett. 5, 213–222. Baselt, D.R., Lee, G.U., Natesan, M., Metzger, S.W., Sheehan, P.E., Colton, R.J., 1998. Biosens. Bioelectron. 13, 731–739. Binasch, G., Grünberg, P., Saurenbach, F., Zinn, W., 1989. Phys. Rev. B 39, 4828–4830. Biswas, A., Panigrahi, R., Pal, M., Chakraborty, S., Bhattacharya, P., Chakrabarti, S., Chakravarty, R., 2013. J. Med. Virol. 85, 1340–1347. Bonino, F., Marcellin, P., Lau, G.K.K., Hadziyannis, S., Jin, R., Piratvisuth, T., Germanidis, G., Yurdaydin, C., Diago, M., Gurel, S., 2007. Gut 56, 699–705. Cai, Z.J., Lou, G.Q., Cai, T., Yang, J., Wu, N.P., 2011. J. Clin. Virol. 52, 288–294. Chen, J., Chen, D., Yuan, T., Chen, X., 2011a. Nano Biomed. Eng. 3, 203–210.
X. Zhi et al. / Biosensors and Bioelectronics 54 (2014) 372–377
Chen, J.S., Yin, J.H., Tan, X.J., Zhang, H.Q., Zhang, H.W., Chen, B.C., Chang, W.J., Schaefer, S., Cao, G.W., 2007. J. Clin. Virol. 38, 238–243. Chen, L., Bao, C.C., Yang, H., Li, D., Lei, C., Wang, T., Hu, H.Y., He, M., Zhou, Y., Cui, D.X., 2011b. Biosen. Bioelectron. 26, 3246–3253. Chen, Y.M., Wu, S.H., Qiu, C.N., Yu, D.J., Wang, X.J., 2013. Braz. J. Med. Biol. Res. 46, 614–622. Duarte, C., Salm, E., Dorvel, B., Reddy, B., Bashir, R., 2013. Biomed. Microdevices 15, 821–830. Edelstein, R.L., Tamanaha, C.R., Sheehan, P.E., Miller, M.M., Baselt, D.R., Whitman, L.J., Colton, R.J., 2000. Biosen. Bioelectron. 14, 805–813. Feng, J., Wang, Y.Q., Li, F.Q., Shi, H.P., Chen, X., 2011. Detection of magnetic microbeads and ferrofluid with giant magnetoresistance sensors. In: Du, Y.W., Judy, J., Qian, Z., Wang, J. (Eds.), 1st International Symposium on Spintronic Devices and Commercialization. Gao, G., Huang, P., Zhang, Y.X., Wang, K., Qin, W., Cui, D.X., 2011. CrystEngComm 13, 1782–1785. Graham, D.L., Ferreira, H.A., Freitas, P.P., Cabral, J.M.S., 2003. Biosens. Bioelectron. 18, 483–488. Grandjacques, C., Pradat, P., Stuyver, L., Chevallier, M., Chevallier, P., Pichoud, C., Maisonnas, M., Trepo, C., Zoulim, F., 2000. J. Hepatol. 33, 430–439. Guan, X., Guo, J., Shen, P., Yang, L., Zhang, D., 2010. Food Anal. Methods 3, 313–320. Guirgis, B.S.S., Abbas, R.O., Azzazy, H.M.E., 2010. Int. J. Infect. Dis. 14, E941–E953. Hepatology, C.S.o, 2011. Chin. J. Epidemiol. 32, 405–415. Hirayama, H., Kageyama, S., Moriyasu, S., Sawai, K., Minamihashi, A., 2013. J. Reprod. Dev. 59, 321–326. Hirayama, H., Kageyama, S., Moriyasu, S., Sawai, K., Onoe, S., Takahashi, Y., Katagiri, S., Toen, K., Watanabe, K., Notomi, T., 2004. Theriogenology 62, 887–896. Horibe, D., Ochiai, T., Shimada, H., Tomonaga, T., Nomura, F., Gun, M., Tanizawa, T., Hayashi, H., 2007. Int. J. Cancer 120, 1063–1069. Hu, H.Y., Yang, H., Huang, P., Cui, D.X., Peng, Y.Q., Zhang, J.C., Lu, F.Y., Lian, J., Shi, D.L., 2010. Chem. Commun. 46, 3866–3868. Hu, H.Y., Yang, H., Li, D., Wang, K., Ruan, J., Zhang, X.Q., Chen, J., Bao, C.C., Ji, J.J., Shi, D.L., 2011. Analyst 136, 679–683. Hussain, M., Chu, C.J., Sablon, E., Lok, A.S.F., 2003. J. Clin. Microbiol. 41, 3699–3705. Ihira, M., Akimoto, S., Miyake, F., Fujita, A., Sugata, K., Suga, S., Ohashi, M., Nishimura, N., Ozaki, T., Asano, Y., 2007. J. Clin. Virol. 39, 22–26. Keeffe, E.B., Dieterich, D.T., Han, S.H.B., Jacobson, I.M., Martin, P., Schiff, E.R., Tobias, H., 2008. Clin. Gastroenterol. Hepatol. 6, 1315–1341. Kim, D., Lee, J.R., Shen, E., Wang, S.X., 2013a. Biomed. Microdevices. 15, 665–671. Kim, D., Marchetti, F., Chen, Z.X., Zaric, S., Wilson, R.J., Hall, D.A., Gaster, R.S., Lee, J.R., Wang, J.Y., Osterfeld, S.J., 2013b. Sci. Rep., 3. Lee, W.M., 1997. N. Engl. J. Med. 337, 1733–1745. Li, Q.C., Fang, J.H., Liu, X., Xi, X., Li, M.J., Gong, Y.F., Zhang, M.Z., 2013. Eur. Food Res. Technol. 236, 589–598. Liu, B., Deng, Y., Qin, B., Li, Z., He, N., 2010. Nano Biomed. Eng. 2, 149–154. Liu, C.J., Kao, J.H., 2013. Semin. Liver. Dis. 33, 97–102. Liu, D.Y., Shi, M., Huang, H.Q., Long, Z.C., Zhou, X.M., Qin, J.H., Lin, B.C., 2006. J. Chromatogr. B—Anal. Technol. Biomed. Life Sci. 844, 32–38. Lok, A.S.F., McMahon, B.J., 2007. Hepatology 45, 507–539. Lu, F.M., Zhuang, H., 2009. Chin. Med. J. 122, 3–4. Marquina, C., de Teresa, J.M., Serrate, D., Marzo, J., Cardoso, F.A., Saurel, D., Cardoso, S., Freitas, P.P., Ibarra, M.R, 2012. J. Magn. Magn. Mater. 324, 3495–3498. Mori, Y., Kanda, H., Notomi, T., 2013. J. Infect. Chemother. 19, 404–411. Mori, Y., Notomi, T., 2009. J. Infect. Chemother. 15, 62–69.
377
Moriya, T., Kuramoto, I.K., Yoshizawa, H., Holland, P.V., 2002. J. Clin. Microbiol. 40, 877–880. Naito, H., Hayashi, S., Abe, K., 2001. J. Clin. Microbiol. 39, 362–364. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T, 2000. Nucleic Acids Res. 28, e63. Olinger, C.M., Jutavijittum, P., Hubschen, J.M., Yousukh, A., Samountry, B., Thammavong, T., Toriyama, K., Muller, C.P., 2008. Emerg. Infect. Dis. 14, 1777–1780. Osterfeld, S.J., Yu, H., Gaster, R.S., Caramuta, S., Xu, L., Han, S.-J., Hall, D.A., Wilson, R. J., Sun, S., White, R.L., 2008. Proc. Natl. Acad. Sci. 105, 20637–20640. Pazienza, V., Niro, G.A., Fontana, R., Vinciguerra, M., Andriulli, A., 2013. Clin. Chem. Lab. Med. 51, 1707–1717. Podesva, P., Foret, F., 2013. Curr. Anal. Chem. 9, 642–652. Poole, C.B., Tanner, N.A., Zhang, Y.H., Evans, T.C., Carlow, C.K.S, 2012. PLoS. Negl. Trop. Dis. 6, e1948. Qiao, J., Wang, J.W., Meng, Q.L., Wang, G.C., Liu, Y.C., He, Z.H., Yang, H.B., Zhang, Z.C., Cai, X.P., Chen, C.F., 2013. Virol. J. 10, 228. Qu, D.F., Zhou, H.Y., Han, J.Z., Tao, S.Y., Zheng, B.L., Chi, N., Su, C.L., Du, A.F., 2013. Vet. Parasitol. 192, 98–103. Qutub, M.O., Germer, J.J., Rebers, S.P.H., Mandrekar, J.N., Beld, M., Yao, J.D.C., 2006. J. Clin. Virol. 37, 218–221. Razumovitch, J., França, K.d., Kehl, F., Wiki, M., Meier, W., Vebert, C., 2009. J. Phys. Chem. B 113, 8383–8390. Sagara, Y., Ohi, Y., Matsukata, A., Yotsumoto, D., Baba, S., Tamada, S., Sagara, Y., Matsuyama, Y., Ando, M., Rai, Y., 2013. Breast Cancer 20, 181–186. Song, Q.Q., Wang, L.X., Fang, R., Khan, M.K., Zhou, Y.Q., Zhao, J.L., 2012. Trop. Anim. Health Prod. 45, 247–250. Song, Y.J., Dai, E.H., Wang, J., Liu, H.J., Zhai, J.H., Chen, C.Y., Du, Z.M., Guo, Z.B., Yang, R.F., 2006. Mol. Cell. Probes 20, 121–127. Stuyver, L., Van Geyt, C., De Gendt, S., Van Reybroeck, G., Zoulim, F., Leroux-Roels, G., Rossau, R., 2000. J. Clin. Microbiol. 38, 702–707. Tatematsu, K., Tanaka, Y., Kurbanov, F., Sugauchi, F., Mano, S., Maeshiro, T., Nakayoshi, T., Wakuta, M., Miyakawa, Y., Mizokami, M., 2009. J. Virol. 83, 10538–10547. Tomita, N., Mori, Y., Kanda, H., Notomi, T., 2008. Nat. Protoc. 3, 877–882. Tran, T.T., Trinh, T.N., Abe, K., 2008. J. Virol. 82, 5657–5663. Valsamakis, A., 2007. Clin. Microbiol. Rev. 20, 426–439. Wang, L., Shi, L., Su, J.Y., Ye, Y.X., Zhong, Q.P., 2013. Gene 515, 421–425. Wang, T.Z., Zhang, Y., Huang, G.L., Wang, C., Xie, L., Ma, L., Li, Z.Y., Luo, X.B., Tian, H., Li, Q., 2012. Sci. Chin.—Chem. 55, 508–514. Yang, H., Chen, L., Lei, C., Zhang, J., Li, D., Zhou, Z.M., Bao, C.C., Hu, H.Y., Chen, X., Cui, F., 2010. Appl. Phys. Lett. 97, 0437021–0437023. Yang, H.I., Yeh, S.H., Chen, P.J., Iloeje, U.H., Jen, C.L., Su, J., Wang, L.Y., Lu, S.N., You, S.L., Chen, D.S., 2008. J. Natl. Cancer Inst. 100, 1134–1143. Yang, J.H., Zhang, H., Chen, X.B., Chen, G., Wang, X., 2013. World J. Gastroenterol. 19, 3861–3865. Yeh, S.H., Tsai, C.Y., Kao, J.H., Liu, C.J., Kuo, T.J., Lin, M.W., Huang, W.L., Lu, S.F., Jih, J., Chen, D.S., 2004. J. Hepatol. 41, 659–666. Zeng, G., Wang, Z., Wen, S., Jiang, J., Wang, L., Cheng, J., Tan, D., Xiao, F., Ma, S., Li, W., 2005. J. Viral. Hepat. 12, 609–617. Zhai, S.L., Liu, C.X., Zhang, Q.D., Tao, C.Y., Liu, B., 2012. Transgenic Res. 21, 1367–1373. Zhang, H., Xu, T., Li, C.W., Yang, M.S., 2010. Biosens. Bioelectron. 25, 2402–2407. Zhi, X., Liu, Q.S., Zhang, X., Zhang, Y.X., Feng, J., Cui, D.X., 2012. Lab. Chip 12, 741–745. Zoheir, K.M.A., Allam, A.A., 2010. Anim. Reprod. Sci. 119, 92–96.
