A quantum dots and superparamagnetic nanoparticle-based method for the detection of HPV DNA

Background The recent advance in nanomaterial research field prompts the development of diagnostics of infectious diseases greatly. Many nanomaterials have been developed and applied to molecular diagnostics in labs. At present, the diagnostic test of human papillomavirus (HPV) relies exclusively on molecular test. Hereon, we report a rapid and facile quantum dots (QDs) and superparamagnetic nanoparticle-based hybridization assay for the detection of (HPV) 16 infections which combines the merits of superparamagnetic nanoparticles and QDs and wholly differs from a conventional hybridization assay at that the reaction occurs at homogeneous solution, and total time for detection is no more than 1 h. Methods The probes were labeled with superparamagnetic nanoparticles and QDs. Sixty cervical swab samples were used to perform a hybridization assay with these probes, and the results were compared with type-specific polymerase chain reaction (PCR) method. Results The statistic analysis suggests that there is no significant difference between these two methods. Furthermore, this method is much quicker and easier than the type-specific PCR method. Conclusion This study has successfully validated the clinical performance of our hybridization assay. The advantages in the time of detection and ease of process endow this method with great potential in clinical usage, especially mass epidemiological screening.


Introduction
Human papillomavirus (HPV) is a small non-enveloped DNA virus that merely infects human squamous epithelial cells. Its genome is a double-stranded circular DNA molecule of 8,000 base pairs (bp) which is divided into three parts, including a segment of about 4,000 bp that encodes proteins mainly involved in viral DNA replication and cell transformation, a segment of about 3,000 bp that encodes the structural proteins of the virus particles as well as a segment of about 1,000 bp that contains the origin of viral DNA replication and transcriptional regulatory elements [1,2]. HPVs can cause a large spectrum of epithelial lesions, primarily benign hyperplasia with low malignant potential such as warts, papillomas, and so forth. Based on epidemiological and molecular evidence, HPV types 16 and 18 were recognized as the high-risk types that were carcinogenic in humans [2,3]. HPV-16 accounts for approximately 50% of all cervical cancers, while HPV-18 is the next most common type and typically is found in from 15% to 20% of squamous cell cancers and in a greater proportion of adenocarcinomas [2][3][4][5][6]. However, cervical cancer is a highly preventable disease when early screening programs are employed that facilitate the detection and treatment of precancerous lesions. Assisted by early detection, the 5-year survival rate for the earliest stage of invasive cervical cancer can be fairly high [7,8].
In recent years, various nanomaterials have been applied to the field of molecular diagnostics [9,10].
Quantum dots (QDs), one of these nanomaterials, are nearly spherical semiconductor particles with diameters from 2 to 10 nm, comprising 200 to 10,000 atoms. QDs have size-controlled luminescence functions, which mean the same material with variable sizes can exhibit different colors under the excitation of an appropriate wavelength; broad absorption spectra; and narrow emission spectra, which mean simultaneous excitation of different colored QDs by a single wavelength [11,12]. In addition, QDs are extremely photostable and highly resistant to photobleaching, which has been reported to be more photostable than a number of organic dyes, including the most stable organic dye, Alexa 488 [13,14]. With their rapid progress, various QDs-bioconjugates have been developed for imaging, labeling, and sensing [15]. Manipulable superparamagnetic nanoparticle through contrived magnetic field is another outstanding nanomaterial, which has been applied to magnetic resonance imaging contrast enhancement, immunoassay, hyperthermia, magnetic drug delivery, magnetofection, cell separation, or cell labeling [16]. Especially in biological separation and diagnosis, the superparamagnetic nanoparticle has a unique advantage over others.
Herein, we report a novel detection method of HPV DNA combining the advantages of QDs and manipulability of superparamagnetic nanoparticles and validate it clinically.

Collection of samples
One hundred sixty cervical swab samples were collected from outpatients at our department, and the written informed consent was obtained. Ten HPV-16-negative and ten HPV-16-positive human DNA samples were kept in the clinical laboratory of our department. QIAamp ® DNA Blood Mini Kits (Qiagen) were used to extract DNA according to the manufacturer's protocol. All DNA samples were eluted with the same volume and then frozen in -70°C until further analysis after quantitated with UV spectrometer (Beckman Coulter, Inc., Beijing, People's Republic of China).

Preparation of CdTe QD-labeled DNA probes
The QD-labeled DNA probes were synthesized according to MY Gao and Dai Zhao [17,18]. In brief, firstly, tellurium powder and NaBH 4 was added into a 100-mL flask with 50 mL of Milli-Q water. The reaction was implemented in room temperature with N 2 protection and lasted until the Tellurium powder disappeared in the flask. Secondly, 86.6 mg of CdCl2 and 79.22 μL of 3-mercaptopropionic acid were dissolved in a threenecked flask with 297 mL of Milli-Q water under N 2 protection. One molar NaOH solution was used to adjust the pH of the mixture to 9.1 under stirring. The NaHTe solution prepared in the first step was added to the reaction mixture under N 2 protection. The resultant mixture was stirred for about 20 min and then boiled at 100°C. The reflux time to get the CdTe QDs was 1 h. X-Ray diffraction (XRD) was used to confirm the crystalline phase of QDs. Four milliliter of CdTe QDs, approximately 100 μg of DNA oligonucleotide second probe described by Lee et al. [19] (Table  1) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) amounting to ten times the mole of DNA, were mixed in 0.05 M Tris-HCl and 0.02 M NaCl buffer (pH 7.2) under room temperature. The resultant product was CdTe QD-labeled probe, and excessive oligonucleotide probes were removed by dialysis against a pH 7.0 PBS buffer using a celluloseacetate membrane. The emission spectrum of resultant QD-labeled probes was characterized by LS 55 luminescence spectrometer (Perkin-Elmer, Beijing, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the conjugation of QDs and probes.

Preparation of superparamagnetic nanoparticle
The superparamagnetic nanoparticles were synthesized according to Nagao et al. with slight modification [20]. Briefly, 5 mL of 2-M FeCl 2 and 20 mL of 1-M FeCl 3 were mixed in 212 mL of Milli-Q water that had been bubbled with nitrogen for 30 min. Fe 3 O 4 nanoparticles were chemically co-precipitated by adding 12 mL of NH 3 solution at room temperature under continuous mixing and washed four times in water and several times in ethanol. During washing, the superparamagnetic Fe 3 O 4 nanoparticles were separated with a NdFeB magnet, and the particles were finally dried in a vacuum oven at 70°C. The transmission electron microscopy (JEOL, Tokyo, Japan) was used to characterize the size of the magnetic nanoparticles. XRD was used to confirm the crystalline phase of superparamagnetic nanoparticles.

Determine of cutoff value and validation of QDs and superparamagnetic nanoparticle-based hybridization
Ten HPV-16-negative human DNA samples were used to determine the cutoff value of QDs and superparamagnetic nanoparticle-based hybridization. The detection procedure was described in detail in the next section ( Figure 1). The cutoff value was defined as the mean fluorescence intensity of HPV-16-negative human DNA samples minus double standard deviations (CV). A result under cutoff value in succedent detection was determined as a positive result. The ten HPV-16-positive samples were used to validate our hybridization assay on the basis of the cutoff value.

Detection of HPV-16 with QDs and superparamagnetic nanoparticle-based hybridization
The rationale of QDs and superparamagnetic nanoparticle-based hybridization is illustrated in Figure 1. A 0.05μg biotin-labeled capture probes and QD-labeled detective probes described by Lee et al. [19] (Table 1) were mixed adequately with 2 μL of DNA samples in a volume with a total of 100-μL-long oligo hybridization solution (Corning Incorporated, Shanghai, China) and Figure 1 The rationale of QDs and superparamagnetic nanoparticle-based hybridization.

Statistical analysis
The comparison between QDs and superparamagnetic nanoparticle-based hybridization and type-specific PCR was analysized by the Statistics Package for Social Sciences (SPSS) software. A p value above 0.05 was considered that there was no significant difference between the two methods.

Characterization of quantum dots
The as-prepared quantum dots are red solution. According to the absorbance spectrum and emission spectrum measured by UV spectrophotometer and luminescence spectrometer, they could be excited effectively under ultraviolet band, and their maximum emission peak is about 530 nm, which means the resultant quantum dots is fluorescence-active and could be used as a fluorescent probe (Figures 2, 3). The X-Ray diffraction analysis indicates that the as-prepared QDs exhibit a zinc blende cubic structure ( Figure 4A). The position and relative intensity of most peaks match well with standard CdTe powder diffraction data (JCPDS82-0474). The SDS-  PAGE results under UV lamp indicate that probes have been conjugated to QDs ( Figure 5A).

Characterization of superparamagnetic nanoparticles
To demonstrate the formation of superparamagnetic nanoparticles, the as-prepared Fe 3 O 4 solution was dropped on the copper grid coated with carbon film and characterized by transmission electron microscopy (JEOL, Tokyo, Japan. As seen in Figure 6, the size of Fe 3 O 4 nanoparticles is about 20 nm. The power XRD pattern also shows that the as-prepared magnetite nanoclusters have an inverse spinel type structure ( Figure 4B). The position and relative intensity of most peaks match well with standard Fe 3 O 4 powder diffraction data (JCPDS89-0688), indicating that the magnetite nanocrystals in nanoclusters are crystalline. In addition, the nanoparticles could be enriched in 2 min by a NdFeB magnet, which means they have good magnetic property. After the removal of external magnetic field, these particles could be easily dispersed, suggesting their paramagnetism. The vibrating sample magnetometer (VSM) results of as-synthesized superparamagnetic nanoparticles indicate that they exhibit superparamagnetic behavior with a saturation moment of about 42.5 emu/g at 300 K, as shown in Figure 7.
The SDS-PAGE results under silver staining indicate that probes have been conjugated to superparamagnetic nanoparticles ( Figure 5B). The cutoff value of QDs and superparamagnetic nanoparticle-based hybridization Ten HPV-16-negtive samples were repeated three times with the abovementioned method; the means were used to determine the cutoff value. According to the data, the cutoff value of this assay was defined as 14.5, any result under 14.5 from the 160 DNA samples was considered as positive one (Figure 3). Based on this cutoff value, all of the ten HPV-16-positve DNA samples were determined as positive results.
Comparison of QDs and superparamagnetic nanoparticlebased hybridization with type-specific PCR The 160 outpatients' DNA samples were checked with QDs and superparamagnetic nanoparticle-based hybridization and type-specific PCR. The results were analyzed with the SPSS software. According to our assay, the infectious rate of HPV 16 in these female outpatients is about 8.1% (13/160) by hybridization method and about 6.9% (11/160) by type-specific PCR method. All samples were detected by DNA sequencing, and the two samples with controversial results were confirmed positive. However, no significant difference was seen between the two methods for analysis of the paired c 2 test (Table 2).

Discussion
In this paper, we have successfully developed a novel and facile hybridization for the qualitative detection of HPV-16 in cervical swab samples. Compared with type-  specific PCR, the greatest advantages of our QDs and superparamagnetic nanoparticle-based hybridization consists in the time of detection and ease of process. Generally speaking, type-specific PCR for detection of HPV-16 DNA takes a skillful laboratory assistant about 4 h, while our hybridization assays only need no more than 1 h. In addition, a typical type-specific PCR assay consists of the extraction of DNA of cervical swab samples, PCR reaction and nucleic acid agarose gel electrophoresis and staining of ethidium bromide, while our hybridization assay method only require extraction of DNA of the samples and simple incubation as well as magnetic separation, which has a good acceptability for any average lab assistant.
With the increasing interest in the development of diverse nanomaterials, many researchers all over the world are pushing the envelope to expand the application of those versatile materials in the field of medicine. Up to the present, numerous nanomaterials have been applied to diagnose infectious diseases such as human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, hepatitis C virus (HCV), hepatitis E virus, herpes simplex virus, and so forth [23][24][25][26][27][28]. Surely, nanotechnology brings new opportunities in diagnostics which allows for the diagnosis of infectious diseases in a sensitive, specific, and rapid format at lower costs than current in-use technologies. As declared by Jain KK, applications of nanotechnology are beginning to show an impact on the practice of conventional medicine; it is bound to continue as hotspot of research for next several decades [28].
In conclusion, we showed a rapid and facile hybridization method for the qualitative detection of HPV-16 DNA in cervical swab samples and successfully validated it in 160 clinical samples. It differs from conventional hybridization assays in such a way that the reaction occurs at homogeneous solution and that of conventional hybridization assay bases on the solid supporter such as polyvinylidene fluoride membrane or  nitrocellulose membrane. Therefore, this method has great potential in clinical usage, especially mass epidemiological screening.