Quick genotyping detection of HBV by giant magnetoresistive biochip combined with PCR and line probe assay

Abstract

Genotyping of human hepatitis B virus (HBV) can be used to direct clinically effective therapeutic drug-selection. Herein we report that a quick genotyping method for human HBV was established by a specially designed giant magnetoresistive (GMR) biochip combined with magnetic nanoclusters (MNCs), PCR and line probe assay. Magnetic nanoclusters of around 180 nm in diameter were prepared and modified with streptavidin, and resultant streptavidin-modified magnetic nanoclusters were used for capturing biotin-labeled hybrid products on the detection interface of the sensor. The gene fragments of HBV’s B and C gene types were obtained by PCR based on a template of B- and C-type plasmids. After gene fragments were hybridized with captured probes, streptavidin-modified magnetic nanoclusters could bind with biotin-conjugated gene fragments, and the resultant hydride products could be quickly detected and distinguished by the GMR sensor, with a detection sensitivity of 200 IU mL⁻¹ target HBV DNA molecules. The novel method has great potential application in clinical HBV genotyping diagnosis, and can be easily extended to other biomedical applications based on molecular recognition.

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1. Introduction

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular cancer (HCC). Approximately two billion people worldwide have been infected with the virus and about 350 million people suffer from chronic infection.¹ In China, HBV infection is highly endemic. A seroepidemiological survey on HBV infection conducted in 2006 showed that the hepatitis B surface antigen (HBsAg) carrier rate was 7.18% in the overall population; accordingly, there are an estimated 93 million HBV carriers, and among them 20 million are patients with chronic hepatitis B.^2,3 Hepatitis B virus (HBV) can be classified into nine genotypes, A through I, based on intergroup divergence of 8% or more in the nucleotide sequence.^3–6 Studies to date have shown that genotype A, B, C and D are found in China, and genotype B (41%) and C (53%) are dominant.⁷HBV genotypes may influence hepatitis B e-antigen (HBeAg) seroconversion rates, mutational patterns in the precore and core promoter regions and the severity of liver disease.⁸ Genotyping may be helpful for clinical diagnosis and effective therapeutic drug selection.

Currently, different reported genotyping methods include direct sequencing,^9,10restriction fragment polymorphism (RELP),¹¹ line probe assay (INNO-LiPA),⁴ multiplex polymerase chain reaction (PCR),¹² real-time PCR,¹³oligonucleotide microarray¹⁴ and enzyme-linked immunosorbent assay for genotype-specific epitopes.¹⁵ However, the above-mentioned methods present various problems such as time-consuming, costliness, complicated procedure, low sensitivity, inferior accuracy and specificity, or difficulty of large-scale application.¹⁶ In order to overcome these shortcomings, we tentatively combine giant magnetoresistance (GMR) with line-probe assay (LiPA) to fabricate a biochip to distinguish Chinese dominant HBV genotypes B and C. The salient advantages of LiPA based on reverse hybridization are high specificity and accuracy in comparison with other methods.^4,17

The resistance of a GMR sensor changes with the magnetic field applied to the sensor, so a magnetically labeled biomolecule can induce a signal. GMR sensors are highly sensitive, portable and give a fully electronic readout.¹⁸ In addition, the GMR sensor is inexpensive and the fabrication is compatible with the current VLSI (Very Large Scale Integration) technology, so GMR sensors can be easily integrated with electronics and microfluidics to detect many different analytes on a single chip.¹⁹ In our laboratory, we have successfully developed a magnetic nanoclusters-based method for HBsAg detection.^20–22 However, it did not have the ability to genotype HBV. Real-time PCR for HBV genotyping is time-saving and has a high throughput; however, the main disadvantages of the method are that it has a lower ability to distinguish among genotypes with proximate melting temperature (Tm) values and the length of amplification for real-time PCR has special requirements. The GMR biosensor we developed combines LiPA with general PCR without limitation of length of amplification. This novel detection method combines a highly sensitive GMR sensor, superparamagnetic nanoclusters (MNCs), high specificity and accuracy of LiPA and universality of general PCR.

2. Materials and methods

2.1 Fabrication of the chip

The GMR multilayers were deposited by dc magnetron sputtering onto 3 inch-Si wafers with the structure of NiFeCo (6 nm)/[Cu (2.2 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.²³ Each chip has eight GMR sensors for detection, including three different probes groups, one blank control group (NTC), and four reference sensors (Fig. 1a). The resistances of sensors are about 3 kΩ for the sensors with the feature line width of 5 μm. The area of each sensor is 100 μm × 100 μm in a zigzag pattern (Fig. 1b). The sensors were covered with a 200 nm thick sputtered SiO₂ passivation layer to protect it from environmental corrosion. A layer of SU-8 photoresist was coated on the chip. A microchannel on the GMR sensor, 300 μm in width and 500 μm in depth, was formed by photo lithography. Finally a polydimethylsiloxane (PDMS) cover of 2 mm in thickness was adopted to encapsulate the microchannel.^24,25

Fig. 1 Conformation of the magnetoresistive biochip: (a) fabrication of the biochip; (b) pattern of the GMR sensor.

2.2 Surface modification of sensor and immobilization of probe

The clean sensor surface was pretreated with a 1% (v/v) solution of aminopropyltriethoxysilane (APTES) in water-free toluene for 2 h at room temperature to introduce amino groups on the surface. To remove most of noncovalently bonded silane, the sensor surface was washed 3 times with toluene. Then, the surface was treated with a 1% (v/v) aqueous solution of glutaraldehyde for 1 h at room temperature, followed by 3 water rinsing steps.²⁶ Three hybridized probe solutions (14 μM) were dropped on the corresponding areas of the sensor for 1 h at room temperature. The NTC region was not modified by any probe related HBV. To quench free aldehyde groups on the surface, the surface was treated with a 20 mM solution of aminoacetic acid in water for 1 h prior to the rinsing step.

2.3 Preparation of magnetic nanoclusters modified with streptavidin

Magnetic nanoclusters of 180 nm or so in diameter were synthesized according to our previous report.^20–22 That is, magnetic nanoparticle clusters (MNCs) were synthesized by a one-pot process in which the concentrations of FeCl₃·6H₂O, citrate acid and polyvinyl alcohol (PVA) were 0.1 M, 0.001 M, and 0.05 M, respectively. The as-prepared MNCs were characterized by SEM, HR-TEM, FTIR and XRD, as well as VSM (Vibrating Sample Magnetometer). Carboxyl groups were present on the surface of prepared MNCs and were directly conjugated with streptavidin, resulting in formation of streptavidin-modified MNCs for further use.

2.4 Probe and target DNA

We designed conserved PCR primers and genotype specific hybridization probes according to reported ref. 4,27–29 and analysis of data of complete genomes from the National Center for Biotechnology Information (NCBI) nucleotide sequence database (GenBank). These primers and probes were produced by Invitrogen Life Technologies (USA). Forward primers and reverse biotinylated primers were used to amplify the conserved domain of the HBV genome. Probe T can hybridise with all genotypes of HBV DNA; probes B and C uniquely hybridize with genotype B and genotype C, respectively, of HBV DNA.

Forward primer: 5′-ggacttctctcaattttctaggg-3′

Reverse primer: 5′-Biotin-tgaggcccactcccata-3′

Probe T: 5′-NH₂-(T)₁₅-atcgctggatgtgtctgcggcgtttt-3′

Probe B: 5′-NH₂-(T)₁₅-cccaaatctccagtcactcaccaacctgttgt-3′

Probe C: 5′-NH₂-(T)₁₅-agcacccacgtgtcctggccaaaatt-3′

The target samples were PCR products amplified from clone plasmidHBV DNA containing the complete genome. These samples gave a better approximation of real biomedical samples and therefore gave a direct test case to demonstrate if the GMR biosensors are suitable for clinical applications. HBV plasmids for HBV genotype B and C were kindly provided by Prof. Y. M. Wen (Fudan University, Shanghai, China).

PCR consisted of total reaction volumes of 25 μL, containing 1 μL (200 IU mL⁻¹) HBV plasmid DNA, 2.5 μL 10× PCR buffer (Mg²⁺ Plus), 2 μL (2.5 mM) dNTP mixture, 0.125 μL (5U μL⁻¹) Taq DNA polymerase (TaKaRa, Dalian, China), 1 μL (10 μM) forward primer, 1 μL (10 μM) biotinylated reverse primer and 17.375 μL sterile deionized water (VEOLIA, ELGA-DI MK2, UK). PCR amplification was performed with a thermal cycler (LongGene, L96C, China) under the following conditions: an initial denaturation at 95 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 53 °C for 30 s and 72 °C for 30 s, and a final polymerization at 72 °C for 5 min.

2.5 Hybridization procedure

We used a reverse hybridization method for HBV DNA detection. The experiment schematic is shown in Fig. 2. The oligonucleotide probes were immobilized on the surface of detection sensors using the chemistry described above (Fig. 2b). The target HBV DNA produced by PCR (Fig. 2a) was then hybridized to the probes (Fig. 2c), finally the streptavidin on the surface of magnetic nanoclusters combined with biotin at the terminal of target DNA (Fig. 2d).

Fig. 2 Principle of genotyping: (a) amplification of target DNA by PCR; (b) immobilization of the probe DNA; (c) hybridization of the analyte DNA; (d) binding of the magnetic microspheres.

The biotinylated products of PCR were denatured at 100 °C for 5 min and chilled on ice for 10 min. Then 1 volume of the products solution was diluted with 3 volumes of hybridization solution (Lab Kit, Shenzhen, China) preheated at 50 °C. The mixture was injected into the channel to incubate at 50 °C for 1 h. The channel was then washed by injecting washing solution 1 (2× SSC, 0.1% SDS) for 3 min and washing solution 2 (0.1× SSC, 0.1% SDS) preheated at 50 °C for 3 min. After hybridization, 1× binding & washing buffer was injected to wash the channel, followed by 5 μg μL⁻¹ streptavidin conjugated magnetic nanocluster solution which was incubated for 15 min at room temperature. Next, the channel was washed with 1× binding & washing buffer for 3 min. Finally, the chip was kept in dry conditions.

2.6 Magnetic signal detection procedure

Because of the demagnetizing effect, GMR sensors did not respond to the vertical magnetic field when the sensor surfaces were free of magnetic nanoclusters, and almost no resistance changes of sensors were observed. The calibration of GMR sensors was performed by adjusting the external magnetic field perpendicular to the sensor surface at first. When no magnetic nanoclusters were on the sensor surfaces, the resistance change of GMR sensor at this time was considered to be a reference signal. When nanoclusters were settled on the surfaces of the sensors, a vertical magnetic field of 230 Oe was applied. The signal output of the GMR sensors were obtained by a digital multimeter (Agilent 34401A, USA) under 0.1 mA direct current owing to the occurrence of magnetic nanoclusters, which mean that the GMR sensors were capable of detecting the target DNA. In our study, negative and positive results are determined by the equation as follows: net value = detection value – reference value.

2.7 Data analysis

All data are presented in this paper as mean result ± S.E. Statistical differences were evaluated using the t-test and considered significance at P < 0.05 level. All figures shown in this article were obtained from three independent experiments with similar results.

3. Results and discussion

As shown in Fig. 1, the designed magnetoresistive biochip was successfully fabricated and was different from previous GMR biosensors, with a microchannel that was used to inject examined samples into the sensor detection area. Its advantage is that very little sample is used for multi-biomarker detection, and after examination, the sample can be collected. More importantly, the biochip can be used repeatedly. We found that 0.1 M PBS solution with 0.5% HCl at 100 °C was quickly used to clean the channel and sensor for 3–5 min, then cold 0.1 M PBS solution was injected into the channel for 5 min, allowing contamination from the previous run to be completely cleaned out so that the biochip can be used for the next examination.

In order to investigate the feasibility of the prepared GMR biochip being used for quick genotyping of HBV DNA, firstly we used B- and C-type plasmids of HBV as templates to obtain the corresponding DNA fragments by PCR; as shown in Fig. 3, obtained gene fragments were 395 bp in length. Secondly, we successfully prepared magnetic nanoclusters of approximately 180 nm in diameter, with superparamagnetic behavior and a saturation moment of 43 em μg⁻¹ at 300 K (Fig. S1, S2 and S3 in ESI†). As shown in Fig. S2 in ESI,† the prepared magnetic nanoclusters were nearly spherical, uniform in size and very stable in PBS solution, no aggregation was observed and there were carboxyl groups on their surfaces. After MNCs reacted with streptavidin, streptavidin-modified MNCs were successfully prepared. In the course of examination, when different oligonucleotide probes captured matched target DNA molecules, the magnetic nanoclusters bound on the surface of the GMR sensor, as shown in Fig. 4a, the quantitative detection results are shown in Table 1. When the net value of the T, B or C region is higher than the value of NTC, the corresponding region is defined as positive. When T and B regions are positive and the C region is negative, this sample is defined as HBV genotype B, shown in Fig. 4b. When T and C regions are positive and the B region is negative, this sample is defined as HBV genotype C, shown in Fig. 4c. Whether B or C region is positive or negative, when T region is negative, this sample is without HBV. Our results fully show that the B and C genotypes of HBV can be clearly distinguished by using our established method.

Fig. 3 Electrophoresis pattern of PCR product: (M) DNA marker; (1) PCR product of B type plasmid; (2) PCR product of C type plasmid.

Fig. 4 (a) SEM images of negative (left) and positive (right) regions of GMR biochip; (b) detecting for genotype B HBV DNA; (c) detecting for genotype C HBV DNA.

Table 1 Resistance changes of sensors for different genotype samples (n = 4, X̄ ± SE)

Genotype samples		Signal change, in mΩ (positive signals in bold)
		NTC region	T region	B region	C region
Genotype B	Reference	32.50 ± 5.90
	Detection	42.50 ± 8.54	262.50 ± 27.80	262.50 ± 14.93	47.50 ± 8.54
	Net value	10.00 ± 2.64	230.00 ± 21.90	230.00 ± 9.03	15.00 ± 2.64
Genotype C	Reference	28.75 ± 3.50
	Detection	42.50 ± 11.08	272.50 ± 27.80	52.50 ± 4.79	203.75 ± 15.19
	Net value	13.75 ± 7.58	243.75 ± 24.30	23.75 ± 1.29	175.00 ± 11.69

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It is well known that 2000 IU mL⁻¹ of serum HBV DNA titer has been used as an important standard to classify different phases of chronic hepatitis B.^3,30,31 Our results show that 200 IU mL⁻¹HBV DNA could be quickly detected and distinguished between different genotypes by the GMR biochip. Therefore, the established GMR biochip can be effectively used for clinical HBV genotype diagnosis, and also can meet the requirement of clinical diagnosis, and therefore has great potential application in clinical HBV genotype diagnosis.

4. Conclusions

In summary, we successfully established a quick, sensitive genotyping method for human hepatitis B virus (HBV) based on a specially designed giant magnetoresistive (GMR) biochip combined with MNCs, PCR and line probe assay. Compared with previous reported methods, our method has obvious advantages as follows:

1. The specifically designed magnetoresistive biochip has a microchannel, which is very convenient to send smaller samples into the examination area and decrease reagent amounts and examination cost, making it particularly suitable for detection of very little amounts of samples.

2. In the detection method, we used magnetic nanoclusters composed of many magnetic nanoparticles to capture the DNA fragment hybridization products. Because the prepared magnetic nanoclusters possess superparamagnetic properties, they markedly decreased the background noise signal, and enhanced the repeatability of examined results.

3. In our method, after hybridization, the detection time of the GMR sensor is only 15 min, which is markedly lower than the traditional 2 h, and hence is a markedly shortened examination time.

In conclusion, we successfully developed a quick, sensitive genotyping method of human hepatitis B virus based on a specially designed giant magnetoresistive (GMR) biochip combined with MNCs, PCR and line probe assay, which has distinct advantages over the traditional GMR detection method, and possesses great potential application prospects in clinical HBV genotype diagnosis.

Acknowledgements

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), and the National Natural Science Foundation of China (Grant No. 60971039).

References

1. World Health Organisation, B Hepatitis, http://www.who.int/mediacentre/factsheets/fs204/en/index.html.
2. F. M. Lu and H. Zhuang, Chin. Med. J., 2009, 122, 3–4.
3. D. Jia and L. J. Li, Chinese Journal of Epidemiology, 2011, 32, 405–415.
4. M. Hussain, C. J. Chu, E. Sablon and A. S. F. Lok, J. Clin. Microbiol., 2003, 41, 3699.
5. T. T. Tran, T. N. Trinh and K. Abe, J. Virol., 2008, 82, 5657–5663.
6. C. M. Olinger, P. Jutavijittum, J. M. Hubschen, A. Yousukh, B. Samountry, T. Thammavong, K. Toriyama and C. P. Muller, Emerging Infect. Dis., 2008, 14, 1777–1780.
7. G. Zeng, Z. Wang, S. Wen, J. Jiang, L. Wang, J. Cheng, D. Tan, F. Xiao, S. Ma, W. Li, K. Luo, N. V. Naoumov and J. Hou, J. Viral Hepatitis, 2005, 12, 609–617.
8. C. J. Chu and A. S. Lok, Hepatology, 2002, 35, 1274–1276.
9. H. Norder, B. Hammas, S. Lofdahl, A. M. Courouce and L. O. Magnius, J. Gen. Virol., 1992, 73, 1201–1208.
10. A. Valsamakis, Clin. Microbiol. Rev., 2007, 20, 426–439.
11. M. Mizokami, T. Nakano, E. Orito, Y. Tanaka, H. Sakugawa, M. Mukaide and B. H. Robertson, FEBS Lett., 1999, 450, 66–71.
12. H. Naito, S. Hayashi and K. Abe, J. Clin. Microbiol., 2001, 39, 362–364.
13. S. Payungporn, P. Tangkijvanich, P. Jantaradsamee, A. Theamboonlers and Y. Poovorawan, J. Virol. Methods, 2004, 120, 131–140.
14. Y. J. Song, E. H. Dai, J. Wang, H. J. Liu, J. H. Zhai, C. Y. Chen, Z. M. Du, Z. B. Guo and R. F. Yang, Mol. Cell. Probes, 2006, 20, 121–127.
15. S. Usuda, H. Okamoto, H. Iwanari, K. Baba, F. Tsuda, Y. Miyakawa and M. Mayumi, J. Virol. Methods, 1999, 80, 97–112.
16. B. S. S. Guirgis, R. O. Abbas and H. M. E. Azzazy, Int. J. Infect. Dis., 2010, 14, E941–E953.
17. S. D. Pas, N. Tran, R. A. de Man, C. Burghoorn-Maas, G. Vernet and H. G. M. Niesters, J. Clin. Microbiol., 2008, 46, 1268–1273.
18. J. Schotter, P. B. Kamp, A. Becker, A. Puhler, G. Reiss and H. Bruckl, Biosens. Bioelectron., 2004, 19, 1149–1156.
19. L. Xu, H. Yu, M. S. Akhras, S.-J. Han, S. Osterfeld, R. L. White, N. Pourmand and S. X. Wang, Biosens. Bioelectron., 2008, 24, 99–103.
20. H. Hu, H. Yang, D. Li, K. Wang, J. Ruan, X. Zhang, J. Chen, C. Bao, J. Ji and D. Shi, Analyst, 2011, 136, 679–683.
21. G. Gao, P. Huang, Y. Zhang, K. Wang, W. Qin and D. Cui, CrystEngComm, 2011, 13, 1782–1785.
22. H. Hu, H. Yang, P. Huang, D. Cui, Y. Peng, J. Zhang, F. Lu, J. Lian and D. Shi, Chem. Commun., 2010, 46, 3866–3868.
23. J. Feng, Y. Wang, F. Li, H. Shi and X. Chen, Detection of magnetic microbeads and ferrofluid with giant magnetoresistance sensors, J. Phys.: Conf. Ser., 2011, 263, 012002.
24. H. Yang, L. Chen, C. Lei, J. Zhang, D. Li, Z. M. Zhou, C. C. Bao, H. Y. Hu, X. Chen and F. Cui, Appl. Phys. Lett., 2010, 97, 043702.
25. L. Chen, C. C. Bao, H. Yang, D. Li, C. Lei, T. Wang, H. Y. Hu, M. He, Y. Zhou and D. X. Cui, Biosens. Bioelectron., 2010, 26, 3246–3253.
26. J. Razumovitch, K.d. Franc,a, F. Kehl, M. Wiki, W. Meier and C. Vebert, J. Phys. Chem. B, 2009, 113, 8383–8390.
27. J. Dong, J. Cheng and Q. Yang, World Chinese Journal of Digestology, 2004, 12, 42–46.
28. C. Grandjacques, P. Pradat, L. Stuyver, M. Chevallier, P. Chevallier, C. Pichoud, M. Maisonnas, C. Trepo and F. Zoulim, J. Hepatol., 2000, 33, 430–439.
29. L. Stuyver, C. Van Geyt, S. De Gendt, G. Van Reybroeck, F. Zoulim, G. Leroux-Roels and R. Rossau, J. Clin. Microbiol., 2000, 38, 702.
30. A. S. F. Lok and B. J. McMahon, Hepatology, 2007, 45, 507–539.
31. E. B. Keeffe, D. T. Dieterich, S. H. B. Han, I. M. Jacobson, P. Martin, E. R. Schiff and H. Tobias, Clin. Gastroenterol. Hepatol., 2008, 6, 1315–1341.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2lc20949g

‡ These authors contributed equally to this work.

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