ARTICLE pubs.acs.org/ac
Ultrasensitive and Selective Electrochemical Identification of Hepatitis C Virus Genotype 1b Based on Specific Endonuclease Combined with Gold Nanoparticles Signal Amplification Shuna Liu, Ping Wu, Wen Li, Hui Zhang, and Chenxin Cai* Jiangsu Key Laboratory of New Power Batteries, Jiangsu Key Laboratory of Biofunctional Materials, Laboratory of Electrochemistry, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, People’s Republic of China ABSTRACT: This work proposes a new strategy for the electrochemical detection of hepatitis C virus (HCV) RNA level and identification of HCV-1b genotype based on the sitespecific cleavage of BamHI endonuclease combined with gold nanoparticles (AuNPs) signal amplification. The assay procedures include the reverse transcription, polymerase chain reaction (PCR) amplification, and electrochemical detection. The samples of 244 mer sequence of HCV RNA from the highly conserved region of HCV-1a, HCV-1b, HCV-1, and HCV-6a, respectively, were first reverse transcribed into complementary cDNA and amplified by PCR. The PCR-amplified samples were then analyzed using a synthetic 21 mer DNA probe, which has been assembled on the electrode surface via a bifunctional molecule of p-aminobenzoic acid (ABA). The results demonstrated that the developed approach can be used for specifically identification of the HCV-1b genotype and selective and sensitive detection of HCV-1b cDNA (244 mer) with a detection limit as low as (3.1 ( 0.8) 1022 M (less than 200 molecules; the concentration refers to the one before PCR amplification). Moreover, the developed method has an ability to discriminate the HCV-1b cDNA sequence from even single-base mismatched DNA sequence, to assay the HCV-1b cDNA level precisely from the mixture of HCV-1, HCV-1b, HCV-1a, and HCV-6a, and to detect HCV in real clinical samples. The protocol has high potential application in molecular diagnostics of HCV in clinical environments.
epatitis C virus (HCV), a single-stranded RNA virus,1,2 displays extensive genetic heterogeneity and is a major causative agent of chronic hepatitis and progressive liver fibrosis leading to cirrhosis and hepatocellular carcinoma.3,4 It infects nearly 3% of the worldwide population, and it is estimated that 30% of patients eventually develop end-stage liver disease.1 At present, the most widely used method for diagnosing HCV is the detection of anti-HCV antibodies based on recombinant proteins from the HCV genome.5,6 However, this assay has huge limitations since it cannot detect viruses during the early stage of infection, at which time antibodies against HCV antigens are not produced. For this reason, the detection of HCV RNA is a valuable alternative assay. The monitoring of HCV RNA in serum or plasma has become an indication for diagnosing or confirming active infections and for assessing the patient response to therapy.1,2 An important task during clinical laboratory diagnosis is the detection of HCV RNA level and of HCV genotype information. A variety of assay methods, including various nucleic acid amplification methodologies,710 electrochemiluminescence,11 surface plasmon resonance,12 piezoelectric sensor,13 an optical method,14 a biosensor based on fluorescence detection,15 electrochemical detection,1618 etc., have been developed for detection of HCV RNA level. In addition, methods such as direct DNA sequencing, restriction fragment length polymorphism, etc., for genotyping HCV have also been developed19 since HCV genotyping is also important for predicting
H
r 2011 American Chemical Society
HCV medical treatment response and treatment duration. Nucleotide sequence analysis is considered to be the reference method for identifying different genotypes of HCV. However, this method is restricted to research settings and is impractical for large-scale clinical studies because it is expensive, time-consuming, and requires special equipment for sequencing.20 Clinically used tests for detecting HCV RNA are mainly based on either qualitative (Amplicor HCV test kit, version 2.0, Roche Molecular Diagnostics) or quantitative approaches (Cobas Amplicor HCV monitor 2.0).21 These approaches cannot analyze the HCV viral level and identify the genotypes of HCV simultaneously. Actually, there is little method available for both evaluating the viral level and identifying the HCV genotyping information at the present time.19 This work develops an electrochemical approach for the assay of HCV RNA level and identification of HCV genotype based on the site-specific cleavage of BamHI endonuclease combined with gold nanoparticles (AuNPs) signal amplification. The strategy is based on the variation of voltammetric signal (Δi) of electroactive label (thionine), conjugated to the 50 -terminus of the synthetic probe DNA via AuNPs, before and after the DNA hybrid was digested by BamHI endonuclease (Figure 1A). The Received: December 3, 2010 Accepted: May 9, 2011 Published: May 09, 2011 4752
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Figure 1. (A) Schematic illustration of the procedures of detection of HCV RNA level and identification of HCV genotype based on the site-specific cleavage of BamHI endonuclease and AuNPs signal amplification. (B) Typical TEM image of the prepared AuNPs. (C) AFM image of AuNPs (1520 nm) used for signal amplification. (D) CVs of a GC electrode in 10 mM PBS (pH 7.4) in the presence (solid lines) and absence (dotted line) of 1 mM ABA at a scan rate of 10 mV/s. (E) CV of AuNPs conjugated to the 50 -terminus of the probe DNA (S1) in 0.1 M H2SO4 solution (10 mV/s). The dotted line is the CV of the S10 -assembled GC electrode after being incubated in the AuNPs solution for 1 h. (F) CV of thionine chemically adsorbed on the surface of AuNPs in 10 mM PBS (pH 7.4).
BamHI endonuclease, a site-specific endonuclease with molecular mass of about 22 kDa,22 recognizes the duplex symmetrical sequence 50 -GGATCC-30 and catalyzes double-stranded cleavage between the guanines in the presence of Mg2þ.23 After digestion by BamHI, the DNA hybrid was cleaved at a specific site and the electrochemical signal of thionine was decreased or disappeared. The extent of the decrease was related to the concentration of target cDNA (244 mer cDNA from HCV RNA genotype 1b (HCV-1b), a clinically relevant analyte) in solution, which forms the basis of the quantitative detection of HCV RNA level. In addition, the identification of the HCV genotype can be achieved based on the developed model. In this study, HCV-1b type was selected to evaluate the application of the established method in the assay of viral loading and identification of HCV type because it has been reported that the patients with HCV are mainly infected by HCV-1b, especially in China.24
’ EXPERIMENTAL SECTION Chemicals. All chemicals and solvents were of reagent grade or better. Hydrogen tetrachloroaurate trihydrate (HAuCl4 3 3H2O), tris(hydroxymethyl)aminomethane (Tris), N-hydroxysuccinimide (NHS, 98%), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide (EDC), and p-aminobenzoic acid (ABA) were purchased from Sigma-Aldrich and used as received. Thionine from SigmaAldrich was recrystallized thrice using water before use. BamHI endonuclease was obtained from Bio-Basic Inc. All solutions were prepared with doubly distilled water.
The synthetic thiol-capped single-stranded 21 mer DNA probe (S1) and 244 mer modified HCV-1b cDNA containing one mismatch base (S3, S4) were purchased from BioSune Biotechnology Co. (Shanghai, China). The probe DNA was designed to hybridize to the highly conserved 50 noncoding region of the HCV genome. The samples of target cDNA were obtained from the highly conserved region from HCV-1a, HCV-1b, HCV-1, and HCV-6a, respectively, by nested reverse transcription polymerase chain reaction (RT-PCR) and processed by the complete Amplicor hepatitis C virus test procedure. The obtained PCR-amplified samples were used for study in this work. The total length of each sample is 244 bases. The concentration was determined by a quantitative assay (Cobas Amplicor HCV monitor 2.0, Roche). The samples were kept in a freezer. The oligonucleotide sequences used in this work are summarized in Table 1. Preparation of Gold Nanoparticles (AuNPs). AuNPs were prepared using the procedures reported previously.25 Briefly, all glassware used in the preparation was thoroughly cleaned in aqua regia (3 parts concentrated HCl, 1 part concentrated HNO3), rinsed in distilled H2O, and oven-dried prior to use. Caution: aqua regia is extremely dangerous and should be handled with extreme caution. Gloves and eye protection are required for handling. In a 1 L round-bottom flask equipped with a condenser, 500 mL of 1 mM HAuCl4 was brought to a rolling boil with vigorous stirring. Rapid addition of 50 mL of 38.8 mM sodium citrate to the vortex of the solution resulted in a color change from pale yellow to burgundy. Boiling was continued for 10 min; the heating mantle was then removed, and stirring was 4753
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Table 1. Sequences of Oligonucleotides Used in This Work sequence (50 to 30 )
description
50 -HS-(CH2)6-AAC GTC GGA TCC CGC GTC
21 mer thiolated probe DNA (S1)
GCC-(CH2)6-NH2-30 0
0
21 mer nonthiolated probe DNA (S1 )
5 -(CH2)6-AAC GTC GGA TCC CGC GTC GCC-(CH2)6-NH2-30 0
244 mer HCV-1b cDNA (S2)
5 -GGC GAC GCG GGA TCC GAC GTT-30
244 mer modified HCV-1b cDNA (1 bp mismatch, S3, the mismatch base, T,
50 -GGC GAC GCG GGT TCC GAC GTT-30
is within the BamHI endonuclease cleavage region) 244 mer modified HCV-1b cDNA (1 bp mismatch, S4, the mismatch base, T, is not within the BamHI endonuclease cleavage region)
50 -GGC GTC GCG GGA TCC GAC GTT-30
244 mer HCV-1a cDNA (7 bp mismatch)
50 -GTT GAC GCG CAA ACC TAC GTC-30
244 mer HCV-1 cDNA (5 bp mismatch)
50 -GTC GAC GCG GAA ACC CAC GTC-30
244 mer HCV-6a cDNA (9 bp mismatch)
50 -GCC GAT GGG GGA TGTTCC GGA-30
continued for an additional 15 min. After the solution reached room temperature, it was filtered through a 0.8 μm Nylon membrane filter. Transmission electron microscopic (TEM) and atomic force microscopic (AFM) images indicated a particle size of 1520 nm (Figure 1, parts B and C). Immobilization of Probe DNA and Conjugation of Thionine. A glassy carbon electrode (GC, 3 mm in diameter, CH Instruments) was used as substrate for assembly of probe DNA. Prior to use, the electrode was polished sequentially with metallographic abrasive paper (no. 6) and slurries of 0.3 and 0.05 μm alumina to create a mirror finish and then sonicated with absolute ethanol and double-distilled water for about 1 min, respectively. It was rinsed thoroughly with double-distilled water and dried under ambient temperature. For assembly of the DNA probe, a monolayer of the bifunctional molecule of p-aminobenzoic acid (ABA) was first formed on the GC electrode surface by cyclic scanning the electrode in a solution of 10 mM phosphate buffer (PBS, pH 7.4) containing 1 mM ABA (six cycles, at the scan rate of 10 mV/s, step a, Figure 1A). ABA presents a single irreversible oxidation peak at ca. 0.8 V (vs SCE, Figure 1D) at a GC electrode in PBS. This anodic peak is ascribed to one-electron oxidation of the amino group turning into its corresponding cation radical,26 which forms a carbonnitrogen linkage at the GC electrode surface resulting the assembly of ABA. The peak current decreases quickly with the successive scanning, indicating the grafting of ABA on the electrode surface (Figure 1D). For molecular diagnostics of human diseases, a short DNA probe is usually terminally anchored or chemically grafted to the solid surface. In this work, the probe DNA (S1) was assembled on the electrode surface by forming the amide between the group of COOH at ABA and the NH2 moiety at the 30 -terminus of S1 via the EDCNHS coupling (step b, Figure 1A). First, the COOH was activated by immersing the ABA-assembled GC electrode into the EDCNHS mixture (5 mM EDC, 10 mM NHS in 10 mM TrisHCl buffer, pH 7.4) for 0.5 h. Then, the electrode was transferred into the solution of S1 (20 μM, 10 mM TrisHCl buffer, pH 7.4) for 3 h. The NH2 moiety at S1 would react with the activated COOH group, resulting to covalently link S1 at the GC electrode surface. The AuNPs were immobilized on the electrode via formation of a AuS bond by incubating the S1-assembled GC electrode in the AuNPs solution for 1 h (step c, Figure 1A). The electrode was thoroughly rinsed with TrisHCl buffer and water, in turn, and stored in the buffer.
The cyclic voltammetric result indicates the characteristic feature of the redox reaction of Au with the oxidation peak at ca. þ1.1 V (vs SCE), corresponding to the formation of Au oxide, and a reduction peak at ca. þ0.9 V, corresponding to the reduction of the Au oxide (Figure 1E, solid line),2729 verifying the immobilization of AuNPs on the electrode. The redox label of thionine was conjugated to the electrode surface by adsorption of thionine on the surface of AuNPs (step d, Figure 1A). This was completed by incubating the electrode in thionine solution for 20 min (1 mM thionine in PBS). The cyclic voltammogram shows a pair of redox peaks at ca. 220 and 270 mV (vs SCE, 10 mV/s, pH 7.4, Figure 1F), which are the characteristic redox features of thionine,3032 demonstrating that thionine has been assembled on the electrode surface via AuNPs. Hybridization and Cleavage. Hybridization was conducted at 37 °C by immersing the thionineS1-immobilized GC electrode in TrisHCl buffer containing the target 244 mer cDNA (S2, 1.0 1011 M) from HCV-1b, HCV-1a, HCV-1, HCV-6a, or synthetic one-base mismatched DNA (S3) for 3 h (step e, Figure 1A). After hybridization, the electrode was thoroughly rinsed with water. BamHI cleavage was performed by incubating the DNA hybrid in TrisHCl buffer (pH 7.4), containing 20 U/μL BamHI, 10 mM MgCl2, and 100 mM NaCl, at 37 °C for 2 h (step f, Figure 1A). After cleavage, the electrode was thoroughly washed and transferred into PBS (10 mM, pH 7.4) to record the electrochemical response. Apparatus. The TEM image was obtained with a JEOL-2010 transmission electron microscope operating at an accelerating voltage of 120 kV. The AFM image was recorded with a Nanoscope IIIa scanning probe microscope (Digital Instruments) using a tapping mode. The samples used for measurements were prepared by casting the AuNPs on the surface of a mica sheet. The solvent was allowed to be evaporated before measurements. The Raman measurement was performed on a LabRam HR 800 UV (Jobin-Yvon) with excitation laser beam wavelength of 514.5 nm. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were employed to sense the assembly of AuNPs and thionine with S1 and to characterize the hybridization and cleavage efficiency since the DPV technique has much higher sensitivity than conventional sweep techniques when detecting very low concentrations of redox probe.33,34 This is achieved by applying a small voltage pulse superimposed on the linear voltage sweep and sampling the differential current at a short time after the pulse. 4754
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Figure 2. Raman spectra of bare (a), ABA-assembled (b), ABAS1assembled (c), ABAS1AuNPsthionine-assembled GC (d), and pure thionine (e).
CV and DPV experiments were performed with a CHI 760B electrochemical workstation (CH Instruments). A two-compartment three-electrode cell with a sample volume of 5 mL was employed. A coiled Pt wire and a saturated calomel electrode (SCE) were used as the counter and the reference electrodes, respectively. The buffer was purged with high-purity nitrogen for at least 30 min prior to each electrochemical measurement, and the nitrogen environment was then kept over the solution to prevent oxygen from reaching the solution. DPV signals were measured using a potential step of 5 mV, pulse width of 25 ms, pulse period of 100 ms, and pulse amplitude of 50 mV.
’ RESULTS AND DISCUSSION The detection of HCV based on site-specific DNA cleavage of BamHI using synthetic oligonucleotide as a probe is illustrated in Figure 1A. To fabricate the sensing platform, one synthetic 21 mer DNA probe (S1) was designed to hybridize to the 50 untranslated region, which is a highly conserved region of the HCV genome for the determination of the HCV-1b. The probe was immobilized on the GC electrode surface with the help of the bifunctional molecule of ABA, which was assembled on the GC electrode via carbonnitrogen linkage formed by cyclic voltammetry.26 The SH group at the 50 -terminus of S1 was conjugated with AuNPs (1520 nm in diameter), and the electroactive label molecule (thionine) was subsequently adsorbed on AuNPs. The aim of using AuNPs is to amplify the voltammetric signals and to enhance the detection sensitivity. Besides the voltammetric characterization (Figure 1DF), the assembly of ABA, S1, and thionine on the electrode surface successively were further characterized and verified by Raman spectra (Figure 2). Two characteristic peaks for carbon materials are observed at ca. 1362 and 1590 cm1 for the Raman spectrum of GC (curve a, Figure 2). After assembly of ABA, several new peaks are found in the range of 830990 cm1 (curve b, Figure 2). They are the characteristic Raman features of a benzene ring,35,36 indicating that ABA has been effectively assembled on the surface of the GC electrode. After probe DNA (S1) was covalently linked with ABA via COOH and NH2 groups, the characteristic Raman peaks of the benzene ring disappear (curve c, Figure 2). These results indicate the assembly of S1 on the electrode surface via ABA. After the adsorption of thionine on the surface of AuNPs, the characteristic Raman peaks of thionine are found at ca. 803, 913, 1035,1234, 1357, 1413, 1446, 1502, 1525, and 1604 cm1 (curve d, Figure 2).37
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These characteristic peak are almost identical with those for pure thionine (curve e, Figure 2). These results demonstrate that ABA, S1, and thionine have been effective assembled on the GC electrode surface. Having characterized the assembly processes, the possibility of detecting HCV-1b cDNA using the developed approach is evaluated. The DPV response of thionineS1 demonstrates an anodic peak at ca. 220 mV (vs SCE, curve a, Figure 3), which is in good agreement with that reported previously.30 After being hybridized with S2, the DPV peak appears at ca. 215 mV (curve b, Figure 3), which has a slightly positive shift comparing that shown in curve a. This shift reflects the effect of the hybridization of S2 on the redox reaction of thionine. However, the height of the peak is almost identical with that shown in curve a, suggesting that hybridization of S1 with S2 does not affect the voltammetric current of the label significantly. After the S1/S2 hybrid was treated with BamHI endonuclease for 2 h at 37 °C, the DPV response of thionine has almost disappeared (curve c, Figure 3) since the label was removed from the electrode surface by the cleavage of BamHI endonuclease at the site of 50 -G/GATCC-30 (Figure 1A). However, after the curve c was amplified, a small and well-defined DPV peak can be clearly observed (curve c0 , Figure 3, please note the different current scales in curves c and c0 ). This peak can be resulted from (i) incomplete cleavage of the thionine-linked S1/S2 hybrid by BamHI, (ii) the assembly of thionine on the surface of nonspecifically adsorbed AuNPs on the electrode surface, (iii) the direct nonspecifically adsorption of thionine on electrode, and/or (iv) the stick of the thionine-linked DNA fragments produced by the cleavage of BamHI. Prolonging the cleavage time (from 2 to 3 h) does not lead to the decrease of the DPV currents (not shown here), indicating that the small DPV peak presented in curve c0 (Figure 3) is not due to the incomplete cleavage of thionine-linked S1/S2 hybrid. To discern the role of those factors, a nonthiolated DNA probe (S10 , 21 mer, 50 -(CH2)6-AAC GTC GGA TCC CGC GTC GCC-(CH2)6NH2-30 ) was designed and immobilized on the GC electrode surface via ABA using the similar procedures to S1 immobilization. After the S10 -assembled GC electrode was incubated in the AuNPs solution for 1 h, the voltammogram did not show the characteristic redox peak of AuNPs (Figure 1E, dotted line), demonstrating the AuNPs cannot be nonspecifically adsorbed on the GC electrode surface. However, after the electrode was incubated in thionine solution for 20 min (1 mM thionine in PBS), a DPV peak is observed for the oxidation of thionine (curve c00 , Figure 3), suggesting the nonspecific adsorption of thionine on the electrode. After the S10 was hybridized with S2 and cleaved by BamHI, the DPV features remain almost unchangeable (not shown here), indicating that the DPV characteristics of the thionine oxidation are not affected by the BamHI although the endonuclease is a biomacromolecule. The height of the DPV peak presented in curve c00 (Figure 3) is almost identical to that depicted in curve c0 of Figure 3, suggesting that the thionine-linked DNA fragments formed by the cleavage of BamHI do not stick to the electrode surface. Therefore, the small DPV peak as shown in curves c and c0 of Figure 3 is due to the direct nonspecifically adsorption of thionine on the electrode. However, the analysis of HCV is based on the variation of the DPV signal of thionine before and after the DNA hybrid was digested by BamHI endonuclease. The slightly nonspecific adsorption of the label does not affect the potential application of the developed method in detection of the HCV-related cDNA. 4755
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Figure 3. DPV responses of thionineS1 in 10 mM PBS (pH 7.4) before (curve a) and after being hybridized with 244 mer cDNA (S2, 1.0 1011 M) from HCV-1b (curve b) for 3 h in 10 mM PBS. Curves c, d, and d0 are DPV responses of thionineS1 after being hybridized with S2 (244 mer cDNA from HCV-1b, curve c), S3 (the synthetic 244 mer modified HCV-1b cDNA containing single mismatch base, curve d; the mismatch base is within the BamHI endonuclease cleavage region), and S4 (the synthetic 244 mer modified HCV-1b cDNA containing single mismatch base, curve d; the mismatch base is not within the BamHI endonuclease cleavage region), respectively, for 3 h in PBS and then digested with BamHI for 2 h at 37 °C in 10 mM TrisHCl buffer (pH 7.4) containing 20 U/μL BamHI, 10 mM MgCl2, and 100 mM NaCl. Curve c0 is curve c displayed in the different current scale (please note the different current scales in curves c and c0 ). Curve c00 depicts the DPV response of thionine nonspecifically adsorbed on the surface of the S10 -assembled GC electrode. Curves eg are the DPV responses of thionineS1 after being hybridized with 244 mer cDNA from HCV-1a (curve e), HCV-1 (curve f), and HCV-6a (curve g), respectively, for 3 h, and then digested with BamHI endonuclease for 2 h. DPV responses were recorded using a potential step of 5 mV, pulse width of 25 ms, pulse period of 100 ms, and pulse amplitude of 50 mV.
To use the developed model in a clinical setting, it is essential to minimize nonspecific binding leading to false positive response. Therefore, two 244 mer modified HCV-1b cDNAs (S3 and S4) were synthesized and used for evaluation of the selectivity of the endonuclease-based analysis method. Each cDNA contains only one mismatch base. The sequences of S3 and S4 are 50 -GGC GAC GCG GGT TCC GAC GTT-30 (S3, 244 mer cDNA, the mismatch base, T, is within the BamHI endonuclease cleavage region) and 50 -GGC GTC GCG GGA TCC GAC GTT-30 (S4, 244 mer cDNA, the mismatch base, T, is not within the BamHI endonuclease cleavage region), respectively. The thionine-conjugated S1 was hybridized with S3 and then treated by BamHI. It is obvious that DPV signal of thionine (curve d, Figure 3) remains almost invariant compared with those presented in curves a and b of Figure 3, suggesting that BamHI has no effect on the S1/S3 hybrid because the hybrid does not contain the specific recognition sequence of the endonuclease. If the S4 was used to hybridize with the thionine-conjugated S1 and then treated by BamHI, the DPV signal of thionine (curve d0 ) had a ca. 20% decrease in comparison with those presented in curves a, b, and d. These results demonstrate that the interaction model can distinguish even single-base mismatched DNA and therefore can be used for high-selectivity determination of HCV-related cDNA. This feature is extremely important for the proposed application in clinical environments. Genetic heterogeneity is a hallmark of RNA viruses in general, and the HCV in particular.38 The high degree of sequence heterogeneity found in HCV isolates requires particularly efficient and specific sequence recognition. However, fabrication of robust probes capable of recognizing a wide range of isolates is a difficult task. Therefore, the specificity is an important parameter in the view of the clinical application. The specificity of the developed method is further evaluated by base-mismatched
cDNA of the 244 mer specific sequences from HCV-1a (7 bp mismatched, 50 -GTT GAC GCG CAA ACC TAC GTC-30 ), HCV-1 (5 bp mismatched, 50 -GTC GAC GCG GAA ACC CAC GTC-30 ), and HCV-6a (9 bp mismatched, 50 -GCC GAT GGG GGA TGT TCC GGA-30 ), respectively. The location of mismatched nucleotides related to the probe varies depending on the heterogeneity at each position of the HCV-1b genome. For better comparison, the thionine-conjugated S1 was hybridized with 244 mer cDNA of HCV-1a, HCV-1, and HCV-6a (each at the concentration of ca. 1.0 1011 M as analyzed by the standard Amplicor hepatitis C virus test), respectively, and then treated with BamHI. It is indicated that no significant change of the DPV signal of thionine in the presence of the mismatched sequences (244 mer cDNA related to HCV-1a, HCV-1, and HCV-6a) was observed (curves e and f, Figure 3) comparing with those depicted in curves a, b, and d in Figure 3, demonstrating that the developed model can be used for specifically distinguishing the HCV genotyping and identifying the HCV-1b. The interaction model of DNABamHI can also be applied to sensitively detect the viral level of HCV-1b. To assess the analytical performance of the proposed model, the thionineS1 was hybridized with various concentrations of 244 mer target cDNA from HCV-1b (S2). The S1/S2 hybrid was then treated by BamHI, and finally, the DPV response of thionine was recorded. The peak currents decrease with the increasing of the concentration of S2 (panel A, Figure 4). The value of Δi, measured as the difference between the peak currents of the thionineS1/S2 hybrid before and after cleavage with BamHI, increases linearly with the level of S2 ranging from ca. 1 1021 to at least 1 1010 M with a correlation coefficient of 0.998 (panel B, Figure 4). The detection limit is estimated to be (3.1 ( 0.8) 1022 M (less than 200 molecules) at a S/N (signal-tonoise ratio) of 3, which is 1 order lower than that obtained using 4756
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Table 2. Measurements of HCV-1b DNA Level in the Mixtures of HCV-1, HCV-1b, HCV-1a, and HCV-6a Using the Developed Methoda added amounts of
Figure 4. (A) Effects of the concentration of 244 mer cDNA from HCV-1b on the DPV response of the thionineS1. The concentration of 244 mer cDNA for the curves a to l is 0, 1 1021, 1 1020, 1 1019, 1 1018, 1 1017, 1 1016, 1 1015, 1 1014, 1 1013, 1 1012, and 1 1011 M, respectively. Before the DPV was recorded, the thionineS1 was hybridized with the 244 mer cDNA (3 h) and then cleaved by BamHI (2 h). (B) The plot of the value Δi on the concentration of 244 mer cDNA. Every point is an average value of five independent measurements.
electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes for detection of cDNA from HCV-1 type (3.71 1020 to 1.82 1021 M).19 Such a low detection limit obtained can be ascribed to the signal amplification of AuNPs. Controlled experiments indicated that the detection limit is ca. 5 1017 M if the label molecule (thionine) was directly conjugated with S1 without use of the AuNPs. Please note that the results presented here are obtained using the PCR-amplified sample (cDNA). We do not try to apply the developed approach to assay the sample without reverse transcription and amplification by the PCR step because the RNA is not stable and because of the complexity of the untreated samples. Different aspects regarding the assay repeatability and precision of the developed method were evaluated. The RSD (relative standard deviation) for assay of 1.0 1015 M 244 mer cDNA from HCV-1b is ca. 2.5% obtained with five different and freshly fabricated detection systems, revealing an acceptable repeatability of the developed method. The assay precision is estimated with the slopes of calibration plots obtained from five independent assay systems. The RSD of these slopes is ca. 3.2%. These results demonstrate the great potential for practical application of the proposed model for not only evaluating the viral level but also identifying the HCV-1b genotyping. Another attractive feature of the developed method is that it can be used for assaying the HCV-1b cDNA level precisely from the mixture of HCV-1, HCV-1b, HCV-1a, and HCV-6a. Four samples of the HCV-1, HCV-1a, and HCV-6a cDNA mixtures with each concentration of 1 1011 M were prepared, and certain amounts of HCV-1b cDNA were added. The amounts of HCV-1b cDNA in the mixtures were measured using the developed method. The results presented in Table 2 indicate that the developed approach has high accuracy in measuring HCV-1b DNA level in the HCV DNA samples. These observations substantially demonstrate that the method can be potentially used for detection of HCV-1b in real clinical samples. The developed method was applied to HCV detection in real samples from sera patients. The sera samples were supplied by a hospital. The real samples containing HCV-1b RNA were subjected to a reverse transcriptase reaction providing cDNA. The HCV-1b cDNA fragment of known sequence was obtained
sample
HCV-1b DNA
measured amounts of
recovery
no.
(M)
HCV-1b DNA (M)b
(%)
11
11
1
1 10
(1.04 ( 0.05) 10
104
2
1 1014
(1.01 ( 0.03) 1014
101
3
1 1017
(0.98 ( 0.08) 1017
98
4
1 1020
(1.05 ( 0.10) 1020
105
a
Each concentration of HCV-1, HCV-1a, and HCV-6a DNA in the mixtures is 1 1011 M. b It is an average value of the five measurements for each sample.
Figure 5. Comparison of the results obtained by the standard Amplicor HCV test (commercial method) and the developed method. The data presented here are the average values of three measurements.
by RT-PCR in the presence of a sense primer from the commercial assay Amplicor hepatitis C virus version 2.0 (Roche Molecular Diagnostics). The obtained amplicons were initially evaluated using the standard qualitative Amplicor HCV test (microwell format with spectrophotometer detection, Amplicor HCV test kit, version 2.0, Roche Molecular Diagnostics) and further tested for hybridization with the S1 probe and cleavage by BamHI using the developed method. The results demonstrate that the samples characterized as positive in the Amplicor test are able to hybridize with S1 and the DPV signal of thionine label decreases significantly after cleavage by BamHI. However, those samples appearing as negative in the Amplicor assay cannot hybridize with S1 and the DPV peak current of the thionine label remains almost unchangeable after cleavage by BamHI. The assay precision of the developed method on the real samples was also evaluated by comparing the results with those obtained using the commercial method. Three PCR-amplified HCV-1b cDNA samples with different concentrations were analyzed by the standard Amplicor HCV test and the presented methods, respectively. The results are depicted in Figure 5. It can be concluded that the results obtained from the two methods are basically the same by considering the experimental errors (within 10%), suggesting the developed approach can be used for HCV detection in the real samples. By comparing with the commercial method, the developed approach can assay the HCV RNA level and identify the HCV genotype simultaneously. However, the detection time of the 4757
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’ CONCLUSIONS In summary, we have described a new method for the detection of the viral level of HCV-1b and identification of HCV RNA genotype based on BamHI endonuclease combined with AuNPs signal amplification. The major advantages of this enzymatic cleavage assay are its high specificity and sensitivity and ease of performance. The developed protocol can be taken as a general method of DNA detection and is expected to be applicable to other type DNA analysis. ’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected],
[email protected].
’ ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (20773067, 20833006, and 20905036), Research Fund for the Doctoral Program of Higher Education of China (20103207110004), the Foundation of the Jiangsu Education Committee (09KJA150001, 09KJB150006, 10KJB150009, and CX09B_307Z), the Foundation of Jiangsu Provincial Key Laboratory of Palygorskite Science and Applied Technology (HPK201005), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. ’ REFERENCES (1) Le Guillou-Guillemette, H.; Lunel-Fabiani, F. Detection and Quantification of Serum or Plasma HCV RNA: Mini Review of Commercially Available Assays. In Hepatitis C: Methods and Protocols, 2nd ed.; Tang, H., Ed.; Humana Press: Totowa, NJ, 2009; Vol. 510, pp 314. (2) Choo, Q. L.; Weiner, A. J.; Overby, L. R.; Kuo, G.; Houghton, M.; Bradley, D. W. Br. Med. Bull. 1990, 46, 423–441. (3) Fujita, N.; Horiike, S.; Sugimoto, R.; Tanaka, H.; Iwasa, M.; Kobayashi, Y.; Hasegawa, K.; Ma, N.; Kawanishi, S.; Adachi, Y.; Kaito, M. Free Radical Biol. Med. 2007, 42, 353–362. (4) Kiyosawa, K.; Sodeyama, T.; Tanaka, E.; Gibo, Y.; Yoshizawa, K.; Nakano, Y.; Furuta, S.; Akahane, Y.; Nishioka, K.; Purcell, R. H.; Alter, H. J. Hepatology 1990, 12, 671–675. (5) Thomas, J. R.; Hergenrother, P. J. Chem. Rev. 2008, 108, 1171–1224. (6) Huang, H.; Chen, Y.; Ye, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 18666–18670. (7) Hollingsworth, R. C.; Sillekens, P.; Van Deursen, P.; Neal, K. R.; Irving, W. L. J. Hepatol. 1996, 25, 301–306. (8) Damen, M.; Sillekens, P.; Cuypers, H. T. M.; Frantzen, I.; Melsert, R. J. Virol. Methods 1999, 82, 45–54. (9) Lee, S. C.; Antony, A.; Lee, N.; Leibow, J.; Yang, J. Q.; Soviero, S.; Gutekunst, K.; Rosenstraus, M. J. Clin. Microbiol. 2000, 38, 4171–4179.
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