Diagnosis of High-Risk HPV

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DNA Modulates the Interaction of Genetically Engineered DNA-Binding Proteins and Gold Nanoparticles: Diagnosis of High-Risk HPV Infection Ju-Yi Mao, Han-Wei Li, Shih-Chun Wei, Scott G. Harroun, Ming-Ying Lee, Hung-Yun Lin, Chih-Yu Chung, Chun-Hua Hsu, Yet-Ran Chen, Han-Jia Lin, and Chih-Ching Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13873 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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ACS Applied Materials & Interfaces

DNA Modulates the Interaction of Genetically Engineered DNA-Binding Proteins and Gold Nanoparticles: Diagnosis of High-Risk HPV Infection Ju-Yi Mao,†,‡,§ Han-Wei Li,§,¶ Shih-Chun Wei,Δ Scott G. Harroun,‖ Ming-Ying Lee,# Hung-Yun Lin,§,˧ Chih-Yu Chung,§,■ Chun-Hua Hsu,# Yet-Ran Chen, ⊥ Han-Jia Lin,*,§ and Chih-Ching Huang*,§,˧,▲ †

Doctoral Degree Program in Marine Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan

‡

Doctoral Degree Program in Marine Biotechnology, Academia Sinica, Taipei 11529, Taiwan

§

Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan

¶

iStat Biomedical Co., Ltd, New Taipei City 22102, Taiwan

Δ

Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan

‖

# ˧

Department of Chemistry, Université de Montréal, Montréal, Québec H3C 3J7, Canada

Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan

Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 20224, Taiwan

■

Ten Giga Bio-Technology, Keelung 20224, Taiwan

⊥

Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan

▲

School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

ABSTRACT: Gene detection has an important role in diagnosing several serious diseases and genetic defects in modern clinical medicine. Herein, we report a fast and convenient gene detection method based on the modulation of the interaction between a heat-resistant double-stranded DNA (dsDNA)-binding protein (Sso7d) and gold nanoparticles (Au NPs). We prepared a recombinant Cys-Sso7d, which is Sso7d with an extra cysteine (Cys) residue in the N-terminus, through protein engineering to control the interaction between Sso7d and Au NPs. Cys-Sso7d exhibited a stronger affinity for Au NPs and more easily induced the aggregation of Au NPs than Sso7d. In addition, Cys-Sso7d retained its ability to bind with dsDNA. The aggregation of Au NPs induced by Cys-Sso7d was diminished in the presence of dsDNA, which could be utilized as a transduction mechanism for the detection of the polymerase chain reaction (PCR) products of human papillomavirus (HPV) gene fragments (HPV types 16 and 18). The Cys-Sso7d/Au NP probe could detect as little as 1 copy of the HPV gene. The sensitivity and specificity of the Cys-Sso7d/Au NP probe for Pap smear clinical specimens (n=52) for HPV 16 and HPV 18 detection were 85.7%/100.0% and 85.7%/91.7%, respectively. Our results demonstrate that the CysSso7d/Au NP probe can be used to diagnose high-risk HPV types in Pap smear samples with high sensitivity, specificity and accuracy. KEYWORDS: genetic engineering, DNA-binding proteins, gold nanoparticles, gene detection; clinical diagnosis; human papillomavirus

INTRODUCTION Cervical cancer is the second most common tumor growth affecting women worldwide, and it is the principal cancer found among women in low- and middle-income developing countries.1 Cervical cancer usually arises from a ring of mucosa called the cervical transformation zone.2 Molecular epidemiological evidence and molecular technology have clearly revealed that certain types of persistent oncogenic human papillomavirus (HPV) infections are the principal (>99%) cause of invasive cervical cancer.3 HPV is a small, non-enveloped, double-stranded

DNA virus and is the most common viral infection of the reproductive tract.4 Most sexually active women and men will be infected at some point in their lives, and some individuals may experience recurrent infections.5 More than 110 different types of HPV have been identified, and approximately 40 different HPVs can infect the genital tract.6 Although most HPV infections are self-limited and are asymptomatic or undetected without any intervention after acquisition, a small proportion of infections with certain types of HPV can persist and progress to cancer.7 Eleven types of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56 and 58) are consistently classified as high risk factors for

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developing cancer.8 However, HPV 16 and 18 infections are the most significant risk factor with respect to its etiology, causing lesions in the early stages.9 If untreated, these lesions may progress to cervical cancer, but this progression usually takes many years.9 As most high-risk HPV infections in women do not develop symptoms, sexually active women are encouraged to routinely undergo screening for identification of infection, type of HPV, abnormal cervical cells and pre-cancerous lesions, starting from 30 years of age.10 A Pap smear coupled with HPV testing is generally used to detect abnormal cervical cells and high-risk HPV in cervical cells to identify early cervical cancer or pre-cancer. The specific detection of highrisk HPV types 16 and 18 is very important because these two types cause most HPV-associated cancers.11 Most specific and sensitive techniques for HPV detection are based on the detection of HPV nucleic acids; these techniques include Southern blotting, in situ hybridization, microarrays, and polymerase chain reaction (PCR).12−16 Conventional DNA amplification using PCR followed by gel electrophoresis can provide precise results; however, its clinical application requires specially trained professionals, reagents, and expensive equipment, and furthermore it is often time-consuming. There is an urgent need for simple, cost-effective, rapid and reliable methods for the molecular diagnosis of HPV that can be employed for field applications, especially in remote areas in underdeveloped countries. Nanoparticles (NPs) have been explored widely as signaling probes for ultrasensitive DNA detection.17 Among them, gold nanoparticles (Au NPs) have been widely employed in many analytical applications for the detection of nucleic acids owing to their outstanding properties, including unique surface plasmon resonance (SPR) with high extinction coefficients and variations in the maximum absorption wavelength upon aggregation, long-term stability, and easy functionalization with a variety of biomolecules.18,19 Recent reports have suggested that Au NPs functionalized with nucleic acids can be effectively used to develop optical and electrochemical assays for virus detection.20−24 Valentini and Poma reported a one-step method for the rapid visual detection of asymmetric PCR products from HIV virus samples based on the controlled aggregation of label-free Au NPs.20 Kumvongpin et al. developed a simple colorimetric assay by using a singlestranded DNA-modified Au NP probe for the detection of the loop-mediated isothermal amplification (LAMP) product of HPV types 16 and 18.21 Azizah et al. employed Au NPs functionalized with single-stranded oligonucleotides for the rapid identification of HPV type 16 genomic DNA coupled with an interdigitated electrode sensor and confirmed by a colorimetric assay.23 Among the existing Au NP-based detection strategies for viruses,20−24 colorimetric assays have attracted considerable attention because of their simplicity, convenience and speed; combined with the relatively inexpensive UV-Vis spectrometers, colorimetric assays are exceptionally suitable for real-time analysis. However, these Au NP probes require the tedious and complicated modification of expensive thiolated oligonucleotides as well as purification of the

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modified nanoparticles.20−23 Therefore, the development of a label-free Au NP-based probe for the specific and sensitive detection of viruses remains an active challenge. Protein-nanoparticle interactions have great potential in many immunological recognition and bio-sensing applications.25 Au NPs are widely used for conjugation with proteins because of their good biocompatibility, easy surface modification, and high protein loading.26 However, the function (activity) of proteins is highly diminished after they are conjugated to Au NPs through covalent or nonspecific physical interactions in most cases.27−30 This phenomenon is due to conformational changes of the protein and even partial unfolding of its structures when it is anchored on a Au NP’s surface. Such protein conformational changes may alter its function or even lead to the exposure of “cryptic” epitopes.31 The activity of proteins on the Au NPs can be mediated by controlling their orientation and density on the particle surfaces.28,31,32 The exposure of active sites and/or bio-recognition sites plays a crucial role in determining the bioactivity of proteins on Au NPs. Recently, many methods have been developed for controlling the orientation of proteins conjugated to Au NPs.33−35 Site-specific methods allow the attachment of proteins in a desired orientation on Au NPs with the bioactive sites (binding epitopes or catalytic sites) freely accessible.28,35−38 For example, Abad et al. developed thioctic acid-capped Au NPs as an excellent building platform for a tailored self-assembled monolayer for specific protein immobilization.37 Reed et al. demonstrated that orientation-controlled ubiquitin and enhanced green fluorescent protein (eGFP) bearing a tetracysteine motif enhanced the specific conjunction between proteins and Au NPs.38 Liu et al. reported that modulation of the activity, density and orientation of pyrophosphatase (PPase) conjugated onto Au NPs can be achieved by adding a cysteine residue to PPase through site-directed mutagenesis.28 In this work, we developed a label-free colorimetric assay based on the modulation of the interaction of Sso7d [a double-stranded DNA (dsDNA) binding protein] with DNA and Au NPs for the rapid and specific detection of HPV 16 and 18 genes (Scheme 1). Sso7d is a small (~7 kDa, 63 amino acids) and abundant chromosomal protein from the hyperthermophilic archaebacteria Sulfolobus solfataricus.39 Sso7d exhibits high thermal (melting point ca. 100 °C), acidic and chemical stabilities, and it binds DNA without marked sequence preference.40 Since Sso7d lacks cysteine residues to mediate the interaction between Sso7d and Au NPs, we engineered a recombinant CysSso7d with an extra cysteine residue in the N-terminal. Cys-Sso7d proteins have similar DNA-binding properties to those of Sso7d. However, we found that Cys-Sso7d could induce greater aggregation of Au NPs (diameter ca. 13 nm) than Sso7d. Furthermore, the Cys-Sso7d-induced aggregation of Au NPs can be controlled in the presence of DNA. The specific detection of HPV operates on the principle that target gene fragments (HPV 16 and 18 types) obtained from PCR decrease the Cys-Sso7d-induced aggregation of Au NPs. Our results demonstrated that this label-free colorimetric Cys-Sso7d/Au NPs probe could detect HPV 16 and 18 with high sensitivity and specificity. 2


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Scheme 1. Schematic representation of DNAdependent Sso7d- or Cys-Sso7d-induced aggregation of Au NPs.

MATERIALS AND METHODS Chemicals and Materials. Phosphate buffer saline (PBS), sodium phosphate, hydrogen tetrachloroaurate hydrate (HAuCl4⋅3H2O), sodium citrate, Ellman’s reagent [5,5ˊ-dithiobis(2-nitrobenzoic acid)], cysteine, cOmpleteTM (EDTA-free protease inhibitor cocktail tablets), glycerol and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). PCR reagents, including GT Premix and PerfectRead polymerase, were purchased from Ten Giga Bio (Keelung, Taiwan). The pET-21a expression vector and Escherichia coli BL21 (DE3) host cells were purchased from Merck KGaA (Darmstadt, Germany). The reagents for recombinant protein preparation, including LB broth, agarose LE and isopropyl β-D-1thiogalactopyranoside (IPTG), were purchased from Bioman (Taipei, Taiwan). Acrylamide liquid (30%), Coomassie Brilliant Blue R-250, 5× SDS-PAGE loading buffer, tetramethylethylenediamine (TEMED) and ammonium persulfate were purchased from MDBio, Inc. (Taipei, Taiwan). Milli-Q ultrapure water (Millipore, Billerica, MA, USA) was used throughout the experiments. Preparation of Recombinant Sso7d Proteins. Based on the primary sequence of Sso7d protein and the codon preference of Escherichia coli (E. coli) BL21 (DE3), the fulllength coding DNA sequences of Sso7d and Cys-Sso7d were chemically synthesized (Figure S1, Supporting Information) and ligated into the pET-21a vector via the NdeI and XhoI sites, respectively (Scheme S1, Supporting Information).41 Then, the recombinant plasmids were transformed into E. coli strain BL21 (DE3) to express Sso7d and Cys-Sso7d recombinant proteins with Cterminal 6× His tags. E. coli cells harboring the recombinant plasmid were cultured at 37 °C with agitation until OD600 = 0.5. The expression of recombinant proteins was

induced by adding 0.2 mM IPTG to the culture media and continued incubation at 14 °C for 20 h. Following cell lysis, the crude extract of cellular proteins was heated at 70 °C for 30 min and then centrifuged at 10,000 × g for 15 min. The 6× His-tagged recombinant proteins in the supernatant were further purified by affinity chromatography on a HisTrap FF column (GE Healthcare Bioscience, Buckinghamshire, UK) according to the manufacturer’s instructions. The affinity chromatography could remove most of the non-recombinant proteins, including heat shock proteins and metallothioneins, even if they are present in the supernatant after heat (70 °C) treatment. The purified proteins were then desalted and concentrated by using Amicon® Ultra Centrifugal Filters (UFC800308, Merck KGaA, Darmstadt, Germany). The protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc, Wilmington, MA, USA) and a NanoOrange® Protein Quantitation Kit (Invitrogen, Carlsbad, CA, USA). The purity and concentration of the proteins were further verified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sso7d- or Cys-Sso7d-Induced Aggregation of Au NPs. Sso7d or Cys-Sso7d (0−400 nM) was incubated with Au NPs (1.0 nM) in sodium phosphate buffer (5 mM, pH 7.4) for 2 h at room temperature. The UV-Vis absorption spectra of Au NPs in the presence of various concentrations of Sso7d or Cys-Sso7d were recorded using a Synergy 4 monochromatic microplate spectrophotometer. The size and morphology of the aggregated Au NPs were observed using transmission electron microscope (TEM; Hitachi HT7700, Tokyo, Japan). Dynamic light scattering (DLS) and zeta potential experiments were conducted using a Zeta sizer Nano-ZS90 analyzer (Malvern Instruments Ltd., Malvern, UK). A TENSOR II Fourier transform infrared (FT-IR) spectrometer (Bruker, Rheinstetten, Germany) was used to analyze the secondary structures of Sso7d or Cys-Sso7d after its adsorption onto Au NPs. Samples were dropped and dried on a ZnSe window and analyzed in the transmission mode. The FT-IR spectra were recorded in the range from 1600 to 1700 cm−1 with 16 scans. High-purity nitrogen was used for purging during the FT-IR measurements to minimize the interference from water vapor. Detection of DNA Using Cys-Sso7d/Au NPs. CysSso7d (100 nM) was incubated with dsDNA (107 bp, 360 bp, 1449 bp or 6213 bp) at concentrations from 1×100 to 1×105 ng mL−1 in 5 mM sodium phosphate buffer (pH 7.4) for 10 min. The mixtures were separately reacted with Au NPs (1.0 nM) in 5 mM sodium phosphate buffer (pH 7.4), then incubated for another 10 min. The UV-Vis absorption spectra of the mixtures were recorded using a Synergy 4 monochromatic microplate spectrophotometer. Clinical Specimens and HPV gDNA Isolation. Cervical scrapings were obtained from ThinPrep cytological test (TCT) samples of 52 females (aged ≧ 20 years) who underwent colposcopic cervical biopsy from June to November in 2015. Among the TCT samples, 19, 7, 12, 9 and 5 were normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion 3



(HSIL) and squamous cell carcinoma (SCC), respectively. Genomic DNA (gDNA) was extracted from the tissue using an iStat Nucleic Acid Extraction Kit (iStat Biomedical Co., Ltd., New Taipei City, Taiwan) in accordance with the manufacturer’s protocol. The gDNA concentration was determined using a NanoDropTM 2000c spectrophotometer (Thermo Fisher Scientific Inc., Rockford, IL, USA). DNA samples were amplified for high-risk HPV typing by multiplex PCR (MPCR) assay and then stored at −20 °C until further use. Detection of HPV genes Using Cys-Sso7d/Au NPs. The detection capability of the Cys-Sso7d/Au NPs probe was examined by coupling it with PCR, using the L1 region amplified by the MY11 and GP6+ primers, the HPV 16 E1 region gene amplified by the PP×16U/F and PP×16U/R primers, and the HPV 18 E1 region gene amplified by the PP×18/F and PP×18/R HPV primers. First, Cys-Sso7d (100 nM) was incubated with the target gene (L1 region, E1 region of types 16 and 18) from clinical PCR samples diluted 1000-fold with sodium phosphate buffer and reacted in 5 mM sodium phosphate buffer (pH 7.4). Then, 1.0 nM of the Au NP probe was mixed with the Cys-Sso7d−dsDNA complex in 5 mM sodium phosphate buffer (pH 7.4) for 10 min. The degree of aggregation of Cys-Sso7d−dsDNA/Au NPs was measured through colorimetric assays using a UV-Vis absorbance spectrometer. Please see the Supporting Information for the details on the synthesis and characterization of Au NPs, determination of the free thiol content, evaluation of DNAbinding activity, displacement of rhodamine B from Au NPs by Sso7d or Cys-Sso7d, determination of binding constant, circular dichroism analysis, and analysis of CysSso7d binding sites onto Au NPs.

RESULTS AND DISSCUSION Expression of Sso7d and Cys-Sso7d. To achieve the goal of modulating the interaction between DNA-binding proteins and Au NPs, a cysteine residue was directly introduced at the N-terminal of Sso7d. Both Sso7d and CysSso7d proteins were heterologously expressed in E. coli. Compared with other cellular proteins in E. coli, Sso7d and Cys-Sso7d were stable at high temperature.39 Therefore, we could remove most contaminant proteins by heating the sample at 70 °C for 30 min before affinity chromatography purification. Figure 1 shows the results of our preparation of recombinant Sso7d and Cys-Sso7d. The SDS-PAGE analysis indicated that the molecular weights of expressed Sso7d and Cys-Sso7d were approximately 10 kDa, consistent with the molecular weight deduced from the cDNA, revealing that Sso7d and CysSso7d were successfully prepared. The final yield of Sso7d and Cys-Sso7d recombinant proteins were 11.1 and 7.5 mg L−1, respectively. The SDS-PAGE analysis also reveals that the purity of expressed Sso7d or Cys-Sso7d after Ni-NTA affinity chromatography purification is higher than 99.5%, and most contaminant proteins from E. coli, which are usually in the range of 20−100 kDa, were removed completely. The free thiol content in Cys-Sso7d was determined using Ellman’s assay and found to be 0.962±0.026

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Figure 1. SDS-PAGE of Sso7d and Cys-Sso7d expression. The proteins were separated on a 12% SDS-PAGE gel and visualized by Coomassie Brilliant Blue R-250 staining.

(mol mol−1 protein; n = 3), suggesting that the sulfhydryl group of the cysteine residue was accessible on the surface of the protein. As a control, the free thiol was undetected in Sso7d. The binding of Sso7d to DNA relies primarily on the lysine-rich residues from site 20 to site 44 of 63 amino acids. The electrophoretic mobility shift assay (EMSA) revealed that Cys-Sso7d possesses almost the same binding capability for DNA, and the saturated binding mass ratio of Cys-Sso7d or Sso7d to dsDNA was approximately 2.0 (Figure S2, Supporting Information). Sso7d and Cys-Sso7d Induced Aggregation of Au NPs. The spherical Au NPs with uniform size (average diameter, 13.1±0.6 nm; from 100 counts) could be simply prepared via the citrate-mediated reduction of HAuCl4. The 13-nm Au NPs undergo obvious color changes upon aggregation as a result of the coupling of the SPR between particles in close proximity, which is highly useful for colorimetric analysis. The citrate-capped Au NPs (13-nm) exhibited a sharp SPR absorption band at 518 nm, which upon aggregation underwent a redshift and decreased, whereas the absorption at 650 nm increased.42 Therefore, the absorbance of Au NPs at 650 and 518 nm (Abs650/518) was used to express the extent of aggregation of Au NPs. Compared with Sso7d, Cys-Sso7d induced larger degrees of aggregation of the Au NPs in sodium phosphate buffer (5 mM, pH 7.4) (Figure 2). The inset in Figure 2 shows that Cys-Sso7d (100 nM) induced substantial aggregation of Au NPs (1.0 nM), which changed the color of the solution from red to purple. The hydrodynamic diameter and zeta potential of citrate-capped Au NPs in sodium phosphate buffer (5 mM, pH 7.4) were determined to be 18±2 nm with a narrow (virtually uniform) distribution and ~−36.5±5.6 mV (n = 5), respectively. The hydrodynamic diameter of Au NPs (1.0 nM) increased significantly to 260±20 nm (n = 5) and 1200±90 nm (n = 5) after incubation with Sso7d (100 nM) and Cys-Sso7d (100 nM), respectively. The negative surface charges on Au NPs were neutralized when Sso7d or Cys-Sso7d was bound to the surface. Thus, the zeta potential of Au NPs decreased to −14.57±3.6 mV (n = 5) and −8.74±2.7 mV (n = 5) after incubation with Sso7d and Cys-Sso7d, respectively. The isoelectric point (pI) of Sso7d is 9.38, and basic amino 4


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Figure 2. UV-Vis absorption spectra of Au NPs (1.0 nM) in the (a) absence and (b, c) presence of (b) Sso7d (100 nM) and (c) Cys-Sso7d (100 nM) in sodium phosphate buffer (5 mM, pH 7.4). Inset: photographic images of the corresponding Au NP solutions.

acid residues such as histidine, lysine, and arginine on the surface of Cys-Sso7d can bind to Au NPs via electrostatic and Au−N interactions in addition to the thiol unit of cysteine having ultrastrong interaction with Au NPs through the formation of the Au−S bond (~180 kJ mol−1).43 In addition to the electrostatic and Au−N interactions and the strong Au−S bond, hydrogen bonding and hydrophobic interactions are likely involved.44 Furthermore, the hexa-histidine units (histidine tags) at the C-terminal of Cys-Sso7d also have strong interactions with the Au surfaces through multivalent Au−N interactions.45 The dissociation constant (Kd) of Cys-Sso7d nanocomposites for Au NPs was determined to be 2.98×10−8 M (Figure S3B, Supporting Information), which is 4.46-fold lower than that of Sso7d (1.33×10−7 M; Figure S3A). The apparent maximal number of binding sites (Bmax) for Cys-Sso7d to Au NPs (~560) is much higher than that of Sso7d (~140) (Figure S3). Moreover, Cys-Sso7d displaces rhodamine B (RB) fluorescent dye from Au NP surfaces faster than Sso7d (Figure S4, Supporting Information). Sso7d or Cys-Sso7d deposited on the surfaces of the Au NPs induce the release of RB molecules into solution, and thus restore the fluorescence of RB. These results suggest that the cysteine at the N-terminal of Cys-Sso7d plays a crucial role in its interaction with Au NPs. The degree of aggregation of Au NPs (1.0 nM) tends to increase with increasing Cys-Sso7d concentration and reaches a plateau at the Cys-Sso7d concentration of 100 nM (curve b in Figure S5, Supporting Information). However, the degree of aggregation of Au NPs was increased upon increasing the concentration of Sso7d from 0 to 150 nM, and was then slightly decreased (curve a in Figure S5). The zeta potential values were higher for Sso7d/Au NP conjugates than for Cys-Sso7d/Au NPs (−5.68 mV vs. −2.36 mV) after Sso7d (500 nM) or Cys-Sso7d (500 nM) was saturated with Au NPs (1.0 nM). As a result, the electrostatic repulsive force coupled with the steric repulsive force (bulky proteins on the surface prevented neighboring Au NPs from attaining the proximity needed to interact and aggregate) led to the Sso7d/Au NPs to retain their higher stability (less aggregation) in solution. However,

the Cys-Sso7d on the surfaces of Au NPs was more likely to exist in flattened structures as a result of the strong interactions (binding) of cysteine and the histidine tags at the N-terminal and C-terminal of Cys-Sso7d (Scheme S2, Supporting Information), respectively. Consequently, the weak steric repulsive force of Cys-Sso7d molecules between the Cys-Sso7d/Au NPs conjugates caused the particles to aggregate easily even if their surfaces are saturated with Cys-Sso7d. Binding Region of Sso7d and Cys-Sso7d to Au NPs. Although the Circular dichroism (CD) spectroscopy and FT-IR spectroscopy analyses reveal that the conformation of Sso7d or Cys-Sso7d did not significantly change after adsorption onto Au NPs (Figure S6 and S7, Supporting Information), we further conducted trypsin digestion coupled with LC-MS/MS analysis to investigate which region of the genetically engineered Sso7d and Cys-Sso7d proteins interacts with Au NPs. The region of Sso7d or Cys-Sso7d adsorbed on the surface of Au NPs ought not to be easily accessible by trypsin due to spatial hindrance, resulting in a low intensity of tryptic peptides in the MS analysis. Therefore, the amino sequences of Sso7d or CysSso7d interacting with Au NPs can be explored according to the quantitative identification of tryptic peptides. Four tryptic peptides from free (unbound) Cys-Sso7d or Sso7d were identified: MATVK, EVDISKIKK, MISFTYDEGGGK, and ELLQMLEK (Figure S8A, Supporting Information). The quantity ratio of ELLQMLEK peptide near the histidine tag is much lower in Sso7d/Au NPs conjugates than in free Sso7d (Figure S8B), suggesting that the histidine tag of Sso7d may be the major region that interacts with Au NPs. However, the quantity ratio of the MATVK peptide is much lower in Cys-Sso7d/Au NPs conjugates than in free Cys-Sso7d (Figure S8B), suggesting that the Nterminal cysteine of Cys-Sso7d may be the other region that interacts with Au NPs. This result supports our reasoning that Cys-Sso7d can bind to particle surfaces by the N-terminal cysteine and the C-terminal histidine tag, leading to easier aggregation of Cys-Sso7d/Au NPs. DNA Mediated the Sso7d- and Cys-Sso7d-Induced Au NP Aggregation. To investigate the DNA-mediated interaction between Sso7d or Cys-Sso7d and Au NPs, the dsDNA (6213 bp) was incubated with Sso7d or Cys-Sso7d and then reacted with Au NPs. Figure 3 shows the Abs650/518 values of Au NPs (1.0 nM) in 5 mM sodium phosphate buffer (pH 7.4) in the absence and presence of Sso7d, Cys-Sso7d, Sso7d/dsDNA and Cys-Sso7d/dsDNA conjugates. The folded Sso7d consists of a doublestranded β-sheet (β1 and β2 strands) and a triple-stranded β-sheet (strands β3, β4 and β5) with a C-terminal αhelix.46 The face of the triple-stranded β-sheet possesses a continuous region of positive electrostatic potential, and Sso7d binds dsDNA by placing this β-sheet across the DNA minor groove, which is independent of the DNA sequence.40 The triple-stranded β-sheet region of Sso7d is anchored to the minor groove of DNA through the insertion of hydrogen bond-donating side chains into the groove and is additionally stabilized by electrostatic and non-polar interactions with the DNA backbone.40 The results showed that Cys-Sso7d−dsDNA complexes in5



Figure 3. Abs650/518 values of (a) Au NPs (1.0 nM) only, (b) − (e) Au NPs in the presence of (b) Sso7d, (c) CysSso7d, (d) Sso7d−dsDNA complexes, and (e) CysSso7d−dsDNA complexes in sodium phosphate buffer (5 mM, pH 7.4) for 10 min. The concentrations of Sso7d, Cys-Sso7d, and dsDNA are 200 nM (1.75×103 ng mL−1), 200 nM (1.77×103 ng mL−1), and 0.1 nM (5.0×102 ng mL−1), respectively. Error bars represent the standard deviations of experiments in triplicate. Inset: photographic images of the corresponding Au NP solutions.

duced a much lesser degree of aggregation of Au NPs than that induced by Cys-Sso7d. This phenomenon can be ascribed to the neutralization of the positive charges on Cys-Sso7d by dsDNA, which prevented the cysteine unit from binding to Au NPs due to the steric effect and electrostatic repulsions. However, the Sso7d−dsDNA complexes induced a slightly higher degree of aggregation of Au NPs than that induced by Sso7d. The TEM images (Figure S9, Supporting Information) show a result consistent with that of the UV-Vis spectra (Figure 3) regarding the induction of different degrees of aggregation of Au NPs by the Cys-Sso7d−dsDNA complexes and Sso7d−dsDNA complexes. Many Cys-Sso7d molecules complexed with one dsDNA molecule may form rigid particle-like structures as a result of possessing neutral charges and of the formation of a disulfide bond between Cys-Sso7d complexes (Scheme 1). However, the Sso7d−dsDNA complexes may be linear and polymer-like. Agarose gel electrophoresis analysis indicated that the Cys-Sso7d−dsDNA complexes show a slightly higher electrophoretic mobility and narrower band width than those of the Sso7d−dsDNA complexes (Figure S2). This result suggests that CysSso7d−dsDNA complexes form a more rigid structure with a smaller radius of gyration than that of linearly structured Sso7d−dsDNA complexes. The linearly structured Sso7d−dsDNA polymer molecule is larger than the Cys-Sso7d−dsDNA complexes in relation to the Au NPs. Thus, a large polymer has several loops that may contain several particles in its coil region.47 As a result, interactions among polymers adsorbed onto many small Au NPs lead to the aggregation (close, irreversible association) and/or agglomeration (loose, reversible association) of the Au NPs (Scheme 1).47 The Cys-Sso7d−dsDNA complex-induced aggregation and the Sso7d−dsDNA complex-induced aggregation of

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Au NPs also exhibited different DNA concentrationdependent behavior. The degree of aggregation of the Au NPs decreased as the concentration of dsDNA increased from 2.44×10−4 nM (1.00 ng mL−1) to 2.44×10−1 nM (1.00×103 ng mL−1) in the Cys-Sso7d−dsDNA/Au NPs system (curve b in Figure S10, Supporting Information). The CysSso7d/Au NPs probe allows detection down to 1.00 ng mL−1 dsDNA with a dynamic range from 1.00 to 1.00×103 ng mL−1. We noted that the degree of Au NP aggregation increased slightly upon increasing the concentration of dsDNA from 1.00 to 1.00×102 ng mL−1 and then decreased in the range of 1.00×102 to 1.00×105 ng mL−1 in the Sso7d−dsDNA/Au NP system (curve a in Figure S10). At a higher concentration of dsDNA (higher ratio of dsDNA to Sso7d), the Sso7d−dsDNA complexes induce less aggregation of Au NPs due to the negative charges of the complexes. The Cys-Sso7d−dsDNA-induced aggregation of Au NPs is also influenced by the length of the dsDNA (Figure S11, Supporting Information). Longer dsDNA conjugated with Cys-Sso7d causes less aggregation of Au NPs because the longer DNA imparts more repulsion and steric effects to the Au NPs compared with shorter DNA. We employed Cys-Sso7d/Au NPs as a colorimetric probe to detect the HPV gene in the following studies as the probe exhibits a consistent DNA dose-response to the aggregation of Au NPs, and the degree of aggregation of Au NPs was highly dependent on the DNA length. We studied the possible interference of protein with the Cys-Sso7d/Au NPs sensor system. The tolerance concentration of bovine serum albumin (within a relative error of ± 5%) for the sensing of dsDNA (360 bp; 1×103 ng mL−1) using the CysSso7d/Au NPs probe was at least 10 µM (Figure S12, Supporting Information). HPV Detection. The HPV detection ability of the CysSso7d/Au NP probe was examined in combination with PCR using the L1 region HPV plasmid DNA, varying from 1 to 105 copies, amplified by the MY11 and GP6+ primers.48 The Cys-Sso7d/Au NP assay detected a minimum of 1 copy in a multiplex PCR (MPCR) assay that combines degenerate E6/E7 consensus primers by monitoring the ratio of absorption coefficients (Abs650/518) (Figure S13A, Supporting Information). The detection limit of our approach is comparable to or better than most Au NP-based probes for the detection of viral genes.20−24 Further, compared to our label-free probe, most of those Au NP-based probes involve tedious processes for labeling and purifying the probes and the use of expensive coupling reagents. Moreover, the detection limit of the Cys-Sso7d/Au NP probe is much better than that of PCR followed by gel electrophoresis (~102 copies; Figure S13B). In addition, the colorimetric Cys-Sso7d/Au NPs probe combined with PCR takes approximately 60 min, whereas conventional gel electrophoresis/PCR combination takes 120 min. We further employed the Cys-Sso7d/Au NP probe for the diagnosis of high-risk HPV from clinical specimens of HPV (n = 52) after PCR amplification of the E1 region gene by the PP×16U/F and PP×16U/R primers and the PP×18/F and PP×18/R primers for HPV 16 and HPV 18, respectively. The Abs650/518 cut-off value of Cys-Sso7d/Au NPs was set at 0.126 and 0.168 (indicated by the dotted 6


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ACS Applied Materials & Interfaces Table 1. Diagnostic performance of the Cys-Sso7d−dsDNA/Au NP assay in the detection of HPV 16 and HPV 18 in Pap smear clinical samples. Cys-Sso7/Au NPs HPV 16

a

a

HPV 18

Multiplex PCR (gold standard.)

Positive Negative Positive Negative

Positive 12 (TP) 2 (FN) 12 (TP) 2 (FN)

Negative 0 (FP) 12 (TN) 1 (FP) 11 (TN)

Sensitivity b (%)

Specificity c (%)

Agreement (%)

85.7

100

92.3

85.7

91.7

88.5

a

TP: true positive, FP: false positive, FN: false negative, TN: true negative. Sensitivity = [TP/(TP + FN)]×100. c Specificity = [TN/(TN + FP)]×100. b

line) for the detection of HPV 16 and HPV 18 (Figure 4). The cut-off value was calculated from the maximum value of the sum of sensitivity ([TP/(TP + FN)]×100) and specificity ([TN/(TN + FP)]×100) from receiver operating characteristic (ROC) curve, which is plotted by sensitivity (yaxis) vs. 1 − specificity (x-axis).49 The sensitivity and specificity of the Cys-Sso7d/Au NP probe with positive clinical specimens for HPV 16 and HPV 18 detection were 85.7%/100% and 85.7%/91.7%, respectively, which are compared with those of PCR (gold standard) (Table 1). The sensitivity and specificity of the Cys-Sso7d/Au NP probe for negative clinical specimens were higher than

Figure 4. Detection of (A) HPV 16 and (B) HPV 18 by Cys-Sso7d/Au NP probe. Gel electrophoresis (Upper panel) and Abs650/518 value of Cys-Sso7d/Au NP probe for the analysis of clinical samples after PCR amplification. Other conditions were the same as the ones described in Figure 3. C, P and S1−S26 represent Au NPs only, Au NPs and Cys-Sso7d, and clinical samples (S1−S26) containing Au NPs and Cys-Sso7d, respectively. Error bars represent the standard deviations of experiments in triplicate.

85.0%. This proposed colorimetric probe revealed high specificity and accuracy for the detection of specific HPV types from Pap smear samples. Although many Au NPbased colorimetric assays have been developed for the detection of viruses, they have rarely been applied to the detection of viruses in complex biological samples.20−24

CONCLUSIONS We have demonstrated that Sso7d and Cys-Sso7d exhibited different interactions with Au NPs and induced aggregation of the particles. The Cys-Sso7d-induced aggregation of Au NPs is highly suppressed in the presence of DNA due to the strong binding ability of Sso7d with dsDNA and to the unique complex structure. Compared to other nanoparticle-based biosensors for DNA analysis, our label-free Cys-Sso7d/Au NP probe does not require a modified complementary probe, and it is applicable for identification of viruses in clinical samples with low interference. Our Cys-Sso7d/Au NPs eliminate the need for sophisticated equipment or the preparation of complicated nanosensors and sample purification. The CysSso7d/Au NP probe coupled with PCR enables the detection of as little as 1 copy of the viral gene. Moreover, the colorimetric Cys-Sso7d/Au NP assay provides high speed, sensitivity, and specificity in the visual detection of highrisk HPV (HPV 16 and HPV 18) in Pap smear samples. This sensing system is simple and cost-effective, and appears to hold great practical potential for the detection of pathogens in biological samples. Aberrant DNA hypermethylation of paired box gene 1 (PAX1) could be used for the detection of cervical intraepithelial neoplasia (CIN) grade 3 and worse lesions (CIN3+) with high sensitivity and specificity. In future work, we will apply the CysSso7d/Au NPs assay to the separate detection of the methylation of PAX1 and HPV 16 and HPV 18 to improve the accuracy of cervical cancer screening.

ASSOCIATED CONTENT Supporting Information Additional information (Scheme S1 and S2 and Figure S1−S13) as noted in the text is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone: +886-2-24622192, ext. 5507. E-mail: [email protected] (H.-J. Lin) 7



*Phone: +886-2-24622192, ext. 5517. Fax: +886-2-24622320. Email: [email protected] (C.-C. Huang)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful to the Ministry of Science and Technology of Taiwan under contracts 104-2113-M-002-008-MY3, 104-2628M-019-001-MY3, 105-2627-M-019-001-MY3, and 105-2622-M019-001-CC2. The assistance of Ms. Ya-Yun Yang and Ms. Ching-Yen Lin from the Instrument Center of National Taiwan University (NTU) for TEM and SEM measurement is appreciated.

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Abstract Graphics:

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Diagnosis of High-Risk HPV

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