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Functional Nanostructured Materials (including low-D carbon)

Preparation and Properties of a Low-Fouling Magnetic Nanoparticles and Its Application to the HPV Genotypes Assay in Whole Serum Lihua Chen, Mingchao Liu, Yan Tang, Chuangfu Chen, Xingxing Wang, and Zhi-Qiang Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04147 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 27, 2019

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ACS Applied Materials & Interfaces

Preparation and Properties of a Low-Fouling Magnetic Nanoparticles and Its Application to the HPV Genotypes Assay in Whole Serum Lihua Chena,b,1*, Mingchao Liua,1, Yan Tangb,1,Chuangfu Chenb, Xingxing Wanga, Zhi-Qiang Hua* a Key

Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science,

Ministry of Education; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, Shandong Province; Key Laboratory of Eco-chemical Engineering; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China. bKey

Laboratory of Prevention and Control of Animal Disease of Xinjiang Corps.

College of Animal Science and Technology, Shihezi University, 832000, Shihezi, Xinjiang, P.R. China. *Corresponding author: Lihua Chen, Zhi-Qiang Hu, E-mail: [email protected], [email protected]; Fax: +86 53284022681; Tel: +86 15054246089 1

These authors contributed equally to this work.

a,bThese

universities contributed equally to this work.

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Gold magnetic particles as a new carrier of disease diagnosis probes have attracted wide attention, but encountered a bottleneck. That is, the interfacial properties of gold magnetic particles are susceptible to the influence of nonspecific biological molecules in actual diagnostic samples. Here, a novel nanoparticle made by the covalent attachment of polyethyleneimine (PEI) and hyperbranched polyether polyol (HPP) onto the gold shell surface of magnetic bead demonstrated not only the low-fouling properties but also the excellent stability in a variety of external environment, especially in complex biological systems. Most importantly, in its application as the probe for sensitive and selective fluorescence detection of high-risk HPV genotypes 18, 16 in buffer, even in 100% serum, a good linear correlation with the concentration of HPV18/16 target DNA ranging from 5 nM to 1 μM was showed with the low detection limits. To our knowledge this is one of few successful examples of direct application of magnetic beads to the detection of disease markers in whole serum, suggesting that this material has good commercial potential and value. Key words: Low-fouling; Gold magnetic particles; HPV genotypes; Fluorescence detection


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1.Introduction The gold magnetic particles consist of both the core of magnetic material and the surface coated with gold shell.1-4 The magnetic core can generate an induced magnetic dipole allowing selective control of location and motion with a magnetic field applied externally. The gold shell layer provides a platform for surface modification, functionalization, tuning magnetic properties and biocompatibility.5 All these characteristics lay its foundation for the clinical diagnostic and therapeutic applications with high sensitivity and selectivity. One case is that Qing Han et al constructed an electrochemical biosensor based on Fe3O4@Au@GSH magnetic sensing film to detect estradiol.6 Another case is that Xuan Xu et al sensitively detected hydrogen peroxide by heme protein functional Fe3O4@Au nanoparticles.7 However, the composition of the actual diagnostic samples is too complex,8,9 including a large number of inorganic substances, organic substances, enzymes, DNA and proteins, which is very easy to change the surface properties of magnetic beads by a strong non-specific adsorption, thus concealing the real test results and leading to erroneous conclusions. Even worse, the magnetic beads may directly miss its all functionality due to the serious agglomeration and precipitation. Therefore, the primary task to achieve this goal of the direct and rapid diagnosis of disease markers with magnetic beads in complex systems, even in whole serum is to enhance the antifouling performance of the magnetic bead interface. Fortunately, the continuous emergence of the new antifouling nanomaterial will provide strong support for circumventing this problem. Until now, poly (ethylene glycol) (PEG)10,11, Polypeptide12,13, poly (carboxybetaine methacrylate)14 15 and phosphorylcholine16,17 have be widely used due to their distinguished properties. But product design involving only these materials is impossible to meet the requirements of increasingly complex detection environment,



so the rhythm of developing new materials and new products has never slowed down. As we known, cervical cancer is one of malignant tumors of genital system threatening women's health and lives. According to the statistics, human papillomavirus (HPV) DNA can be detected in the vast majority of cervical cancer tissue, and thus, the principal etiologic agent, HPV, is considered as a high-risk factor for cervical cancer.18 It is undeniable that most of these deaths could be prevented with proper screening for precancerous lesions or the presence of HPV followed with standard clinical interventions. Unfortunately, a sustained increase in incidence and mortality rate indicated a low screening efficiency of existing technology. Traditional diagnosis based on the cytology (Pap smear or Thin Prep) alone on clinician-obtained samples is well recognized because of its high false negative rate (also was 50% sensitivity). The implementation of HPV-DNA test seems to bring a dawn to the diagnosis of cervical cancer, which is more sensitive in detecting high-risk lesions. However, the existing technology for HPV test can’t get rid of the complex pretreatment and the requirement of professional executor and expensive instrument. Therefore, a novel technical recommendation has to be continually evolved to keep pace with improvements in HPV testing. Here, a novel gold magnetic particle was constructed based on Polyethyleneimine (PEI)19,20 and hyperbranched polyether polyol (HPP)21, which demonstrated the excellent dispersibility, stabilization, biocompatibility, especially a good antifouling performance. Although the main carcinogenic HPV types are more than twelve kinds, HPV genotype 18, 16 account for more than 73%.22 Therefore, in our cases, the specific DNA fragments terminated with NH2 only representing HPV18/16 are designed and anchored on Fe3O4@Au@PEI@HPP NPs with 1,4-butanediol diglycidyl ether (BDE)


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liner. More encouragingly, based on a fluorescent dye, Hoechst 33258, a series of fluorescence experiments on the rapid and direct diagnosis of HPV genotype 18, 16 in buffer or in 100% serum with Fe3O4@Au@PEI@HPP NPs-dependent probes have been successfully carried out. For HPV18 or HPV16 probes, no cross-reactivity with other HPV genotypes was observed. Moreover, a good linear correlation with the various concentration of HPV target DNA in buffer or in 100% serum was demonstrated with the low detection limit, which means that this novel nanoparticle has good practical application ability and great commercial potential.

2 Experimental 2.1. Chemical and materials All oligonucleotide sequences were synthesized and purified by HPLC at Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The sequences of the oligonucleotides are listed in table S1. Hoechst 33258 was also purchased at Shanghai Sangon

Biotechnology

Co.

Ltd.

(Shanghai,China)

Gold

chloride

hydrate

(HAuCl4·XH2O), sodium chloride, polyethyleneimine (PEI) (molecular weights of 18000g·moL-1.),

meso-Erythritol(Ery)

and

bovine

serum

albumin

(BSA,MW:66.43kDa) were supplied by Aladdin Reagents (Shanghai,China). Iron(III) chloride hexahydrate (FeCl3·6H2O), iron(II) chloride tetrahydrate (FeCl2·4H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. 1,4-butanediol diglycidyl ether (BDE) was obtained by Sigma-Aldrich. All other chemicals were analytical grade quality and were used without further purification. All aqueous solutions were prepared with pure water produced by a Milli-Q water purifying system. All experiments were performed at room temperature.



2.2. Apparatus Transmission electron microscopy (TEM) was performed with a JEM-2100 TEM instrument (Hitachi High-Technology Co., Ltd. Japan). Scanning electron microscopy (SEM) was fulfilled with a JEOL JSM-7500FSEM instrument (Hitachi HighTechnology Co., Ltd. Japan) to characterize nanostructures and morphologies of different magnetic NPs. Fourier transform infrared spectroscopy (FTIR) was performed with the Bruker Tensor 70 spectrometer (Bruker Optics, Germany). Contact angle measurements (CA) were carried out using a JC2000C1 goniometer (Shanghai Zhongchen instrument Co., Ltd. China). Magnetic properties of all samples were measured with vibrating sample magnetometer (VSM) (equipped with physical property measurement system (PPMS)) with a maximum applied continuous field of 15,000 Oe at room temperature. All the florescence measurements were carried out with FL-2700 fluorescence spectrophotometer (Shimadzu, Tokyo, Japan). 2.3 The preparation of Fe3O4@Au@PEI@HPP NPs Fe3O4@Au NPs were synthesized by chemical co-precipitation methods as previously reported.23,24 Then the freshly-prepared Fe3O4@Au NPs (5 mL, 2 mg·mL−1) were mixed with equal volume of PEI (20 mM) and stirred for 48 h at 4℃. After incubation of two days, free PEI was removed by washing Fe3O4@Au@PEI NPs several times with water. Subsequently, Fe3O4@Au@PEI NPs were dropped into a mixture containing Ery (0.4 moL·L−1) and BDE (0.1 moL·L−1) in buffer (PH=4) for 24 h at room temperature. Finally, the successfully-prepared Fe3O4@Au@PEI@HPP NPs were collected and washed with water and then re-dispersed in 2.5 mL deoxygenated water for further use (1 mg/mL). 2.4The anti-fouling property of Fe3O4@Au@PEI@HPP NPs


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Here, the nonspecific biomolecule adsorption of Fe3O4@Au@PEI@HPP NPs was measured or monitored with DPV module of CHI660E (the detail procedures were shown in Fig.S1). Firstly, 10 μL Fe3O4@Au@PEI@HPP NPs were dropped on the MGCEs, then, dried in a constant-temperature oven at 37°C for 1 h. Subsequently, the DPV responses of Fe3O4@Au@PEI@HPP NPs modified electrodes were recorded and compared before and after incubation with different samples for 1 h, including BSA (10 mg·mL−1, dissolved with PBS solution), DNA solutions (10-6 M, dissolved with PBS solution) and serum (100%). 2.5 The stability of Fe3O4@Au@PEI@HPP NPs under the various conditions. To study the stability of Fe3O4@Au@PEI@HPP NPs under various conditions, Fe3O4@Au@PEI@HPP NPs (0.2 mg mL-1) were dispersed in different pH (pH 3.0, 5.0, 7.0, 9.0, 11), temperature (-4°C, 37°C, 70°C), salt ion concentrations (0 mM, 1 mM, 10 mM, 50 mM) for 12h, only at 100°C for 5 min. In addition, the potential application of Fe3O4@Au@PEI@HPP NPs in complex media was also evaluated through the incubation of Fe3O4@Au@PEI@HPP NPs with 10%, 50% and 100% serum (V/V) for 12 h. Finally, the stability and dispersion of Fe3O4@Au@PEI@HPP NPs after incubation were studied by naked-eye observation and UV-visible spectrophotometer. Additionally, all results should be compared with the standard sample (Fe3O4@Au@PEI@HPP NPs dispersed in water and stored at 4 °C). 2.6

Attachment

of

specific

gene

on

HPV

genotype

18,

16

onto

Fe3O4@Au@PEI@HPP NPs Specific gene (20 µM, 100 µL) on HPV genotype 18 or 16 and BDE (20 µL) were firstly mixed with Fe3O4@Au@PEI@HPP NPs (100 µL) in solution (pH=4) for 12 h, then magnetic beads were thoroughly washed with 10 mM Tris-HCl (pH=8) and stored at room temperature for further use (Fe3O4@Au@PEI@HPP@probe NPs).



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2.7 Selectivity of Fe3O4@Au@PEI@HPP@probe NPs To investigate the specificity of Fe3O4@Au@PEI@HPP@HPV18 NPs, DNA sequences (Completely complementary sequence: HPV18-CS, Completely noncomplementary sequence: HPV18-NCS) and sequences of disturbing DNA (noncomplementary sequence: HPV16-CS and HPV16-NCS) were respectively prepared in Tris-HCl solution (10 mM, pH=8.0), then Fe3O4@Au@PEI@HPP@HPV18NPs were separately incubated in the above DNA solution for 15 min. After washing three times, 10 μL 1 mg/mL Hoechst 33258 was dropped and the fluorescence signal of the mixture was directly recorded. Simultaneously, four samples (Fig.S7, control 1 (Tris-HCl), control

2

(Tris-HCl+Hoechst33258),

HCl+Fe3O4@Au@PEI@HPP@HPV18

NPs),

control

3

(Tris-

control

4

(Tris-

HCl+Fe3O4@Au@PEI@HPP@HPV18NPs+Hoechst33258)) were prepared for the control experiment. For Fe3O4@Au@PEI@HPP@HPV16 NPs, the essentially consistent process is repeated, except that HPV16-CS and HPV16-NCS replaces HPV18CS andHPV18-NCS as completely complementary sequence and completely noncomplementary sequence and HPV18-CS andHPV18-NCS were considered as the interference sequence, conversely. The detail fluorescent parameters were as follows: excitation slit is10 nm, the width of emission slit is 5 nm and the wave length of the excitation light is 346 nm. The wave length of the emission light is 400-600 nm. 2.8 DNA sensing on Fe3O4@Au@PEI@HPP@probe NPs in buffer or in whole serum 10 µL Fe3O4@Au@PEI@HPP@Probe NPs were directly added into the different concentrations of HPV target DNA (PH=8, diluted in Tris-HCl buffer) one by one for 15 min at 37°C, then, magnetically separated, washed three times and re-dispersed with Tris-HCl buffer. Next, after the addition of 10 μL Hoechst 33258, the fluorescent peaks


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were measured. Finally, all experimental procedures were repeated again, except for some minor changes. That is to say, HPV target DNA in buffer is replaced by HPV target DNA in whole serum. Notably, in whole serum, the control sample was prepared as follows: 10 µL Fe3O4@Au@PEI@HPP@Probe NPs should be incubated only in whole serum for 15 min firstly, then washed three times, finally dispersed in Tris-HCL without (Fig.S7, Control 5) or with (Fig.S7, Control 6) 10 μL Hoechst 33258. 3.Results and discussion 3.1 The choice of materials and the modified chemistry of Fe3O4@Au@PEI@HPP NPs The design of magnetic beads with the excellent dispersibility, stabilization, biocompatibility, efficient target recognition, especially antifouling surfaces has become the key factor of the whole implementation plan, thus, the utilization of diverse coatings and conjugation chemistries is indispensable to improve the performance of magnetic beads. Among them, material selection is the top priority. Here, the synthesis of gold coated iron nanoparticles (Scheme1, (1), Fe3O4@Au NPs) is of special interest, since gold coating results in the formation of air-stable as well as acids and bases stable nanoparticles, which are protected from oxidation and corrosion (the detail characteristics are shown in Fig.S2). Then, based on two key factors, namely, the formation of steric or electronic repulsion among particles for the resistance of particles agglomeration and that of hydration layer on surface of particles for the resistance of protein adsorption, the material with the rich isotropic charge and the hydrophilic properties becomes the ideal candidate. Poly(ethylene glycol) (PEG), polypeptide, poly(carboxybetaine methacrylate) and phosphorylcholine are attracting more attention due to their distinguished properties, but the involvement of the thermal



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instability and sensitivity toward oxidation, expensive prices and the complex chemical modification forced us to abandon these materials. Polyethyleneimine

(PEI),

a

water-soluble

polymer

produced

by

the

polymerization of ethyleneimine, is not a completely linear polymer but a partially branched polymer comprising primary, secondary and tertiary amines. This material has been widely used in bead modification due to its abundant positive charge, low cost and properties that it can undergo a variety of chemical modifications. Therefore, PEI was firstly fixed on gold surface by N-Au bond (Scheme1, (2), Fe3O4@Au@PEI NPs).25,26 As we known, it is very difficult to try to endow the magnetic beads with all the excellent performance only with a single component modification. So erythritol (Ery) was employed to modify on Fe3O4@Au@PEI NPs by a homologous bifunctional crosslinker (1,4-butanediol diglycidyl ether, BDE). The epoxy functional group at one end of BDE reacts with amino groups of PEI by ring-opening reactions in acidic environment, and the other with hydroxyl group of erythritol. Ultimately, hyperbranched polyether polyol (HPP) with the plentiful C-O-C and -OH functional groups

were

generated

on

Fe3O4@Au@PEI

NPs

(Scheme1,

(3),

Fe3O4@Au@PEI@HPP NPs), which is benefit to not only the resistance to external proteins but also steric stabilization on magnetic nanoparticles, due to massive formation of the hydration layer on surface of rich C-O-C groups27. But there is a contradiction that the formation of excess C-O-C will reduce the number of -OH conjugated with probe DNA for further experiment. The best ideal result is that good antifouling performance and biocompatibility were demonstrated at the same time. In fact, the content of C-O-C and OH functional groups is related to the


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ratio of raw material (Ery:BDE). According to our results (Fig.S3), Ery:BDE=4:1was employed. Next, the specific DNA fragments terminated with NH2 only representing HPV18 and HPV16 are designed through the NCBI blast program and modified on Fe3O4@Au@PEI@HPP

NPs

with

BDE

liner

(Scheme1,

(4),

Fe3O4@Au@PEI@HPP@Probe NPs). Notably, the specific sequences of HPV18/16 contain a large number of A:T- base pairs. As we known, Hoechst 33258, a fluorescent dye, can be selectively bound to A:T sequences of minor groove of dsDNA by hydrophobic force, electrostatic interactions, van der Waals interactions and hydrogen bonds, thus emit strong fluorescence at 450 nm with a synergistic effect of these forces28. Interestingly, for ssDNA, only a very weak fluorescence will be demonstrated. Therefore, for complete matching sample, the strong fluorescence intensity will be obtained, which is defined as a positive result (Scheme1, (5)), then the opposite is negative. 3.2 Characterization of Fe3O4@Au@PEI@HPP NPs The morphology, size and structure of Fe3O4@Au@PEI@HPP NPs were successfully characterized by TEM (Fig.1C,1D), SEM (Fig.1G) and FTIR (Fig.1H, curve b). TEM and SEM: As shown in TEM images of Fe3O4@Au@PEI@HPP NPs (Fig.1D), it displayed uniformly spherical structure with a mean diameter of approximate 50 nm which can be further certified from DLS (Fig.1E) and dispersed well with no signs of aggregation. From the SEM image, we can clearly see that after modification of HPP, the particle size of Fe3O4@Au@PEI@HPPNPs (Fig.1G) is obviously larger than that of Fe3O4@Au@PEI NPs (Fig.1F).Interestingly, under HR-



TEM of Fe3O4@Au@PEI@HPP NPs (Fig.1C), around 3 nm polymer film is clearly displayed in the outer layer of gold magnetic particles, which indirectly proves the complex matrix including PEI and HPP may be successfully formed onto gold surface (EDS spectrum of Fe3O4@Au@PEI@HPP NPs is shown in Fig.S4). FTIR: The structure of Fe3O4@Au@PEI@HPP NPs contains a large number of CH2, -NH2, -OH and C-O-C. As shown in Fig.1H (Curve a), the peak at 3435 cm−1 is attributed to -NH2 stretching. The peak at 1634 cm−1 is caused by in-plane bending vibration of -NH2 group. The peaks at 2921 cm−1 and 2877 cm−1 are ascribed to -CH2. These mean that PEI is successfully modified on the surface of gold magnetic particles. After the synthetic HPP was successfully coated on the surface of the nanoparticles, significant increases of the infrared absorption peak at 2921cm-1, 2877cm-1 are observed, which is attributed to the fact that Fe3O4@Au@PEI@HPP NPs contains more -CH2 than Fe3O4@Au@PEI NPs. As for the obvious increases at 3435cm-1 and 1634cm-1, the most reasonable explanation is that the infrared absorption peaks from the newly-formed hydroxyl group on bead and from previous amino group basically overlap. Those new infrared absorption peaks at 1404cm-1, 1085cm-1, 1046cm-1, which are the stretching vibration peak and bending vibration peak of C-O-C, may only come from Fe3O4@Au@PEI@HPP NPs (Fig.1H, curve b). Considering the above two points, we believe that HPP has be successfully synthesized on the surface of gold magnetic particles, which is consistent with the result from TEM and SEM. By the way, Fe3O4@Au@PEI@HPP NPs also have a good magnetic property (VSM curve, Fig.1A) and magnetic response (Fig.1B, without external magnetic field (a) and in external magnetic field (b)). 3.3 The stability of Fe3O4@Au@PEI@HPP Excellent magnetic beads must be guaranteed not to agglomerate in various


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external environments. Therefore, the stability of Fe3O4@Au@PEI@HPP NPs is investigated by the incubation in buffer with different pH, temperature, salt ion concentrations and serum concentrations (Fig.S5). Interestingly, only under different salt

ion

concentrations

and

serum

concentration,

UV

absorption

of

Fe3O4@Au@PEI@HPP NPs has a slight trend of gradual decline (Fig.S5B, S5D). In addition, under pH=11 and 100°C, the UV absorption peak of Fe3O4@Au@PEI@HPP NPs also has a negligible change (Fig.S5A, S5C). However, the color of Fe3O4@Au@PEI@HPP NPs judged by naked eyes is really no obvious difference each other, in all conditions. Therefore, only according to these tiny data fluctuations, it has no sufficient reason to bring a negative impact on the wide application of this novel magnetic bead. 3.4 Antifouling property of Fe3O4@Au@PEI@HPP NPs The antifouling performance of the magnetic bead interface will affect its accuracy and selectivity in the application of disease marker detection. In general, the more hydrophilic the interface is, the better the antifouling efficiency is. The static water contact

angle

values

of

Fe3O4@Au

NPs,

Fe3O4@Au@PEI

NPs

and

Fe3O4@Au@PEI@HPP NPs were recorded, respectively. As we expected, the surface of Fe3O4@Au@PEI@HPP NPs (16.34°, Fig.S6C) are more hydrophilic than that of Fe3O4@Au@PEI NPs (38.30°, Fig.S6B) or Fe3O4@Au NPs (80.81°, Fig.S6A), which implies that Fe3O4@Au@PEI@HPP NPs may have more excellent ability to resist biomolecules adsorption. To verify the above conclusion, 1 μM DNA, 10 mg/mL BSA and 100% serum were employed to evaluate the anti-fouling ability of Fe3O4@Au NPs, Fe3O4@Au@PEI NPs and Fe3O4@Au@PEI@HPP NPs by electrochemistry test. As shown in Fig.2, among three modified electrodes, Fe3O4@Au@PEI@HPP NPs modified electrode has



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minimal change in DPV, even if in 100% serum, the change of DPV (I%=(Ip0-Ip)/Ip0, Fig.S1C) was only about 17%. This means that this material can be applied in 100% serum, which is consistent with the conclusion discussed above. 3.5. Selectivity and the stability of Fe3O4@Au@PEI@HPP@Probe NPs The criterion for Fe3O4@Au@PEI@HPP@Probe NPs with a good application value is the diagnosis conclusion based on the NPs can’t be disturbed by non-specific HPV-DNA in diagnostic samples. In our case, control experiments were firstly carried out to ensure low fluorescence signal interference from all raw materials themselves. As shown in Fig.S7A, fluorescence intensity of four control samples described in section 2.6 is very weak, which implies that the interference of buffer, Fe3O4@Au@PEI@HPP@HPV18 NPs and Hoechst 33258 can be directly neglected. Then, to evaluate the selectivity of Fe3O4@Au@PEI@HPP@HPV18 NPs, HPV18-CS, HPV18-NCS, HPV16-CS and HPV16-NCS were employed one by one. As shown in Fig.3, an obvious fluorescence response (F-F0=243) was shown only in HPV18-CS, around 22 in HPV18-NCS, 31 in HPV16-CS and 24 in HPV16-NCS. This high selectivity to HPV18-CS might be attributed to the high affinity and good selectivity of the probes against HPV18-CS. Moreover, the low response to HPV16-CS indicated the it can be applied

to

the

detection

of

HPV

genotypes.

Fortunately,

Fe3O4@Au@PEI@HPP@HPV16 NPs also demonstrated a good selectivity against HPV16-CS in our cases (Fig.S8). Here, F0 represents the fluorescence value of control 4 in Fig.S7, while F is the fluorescence value after the addition of HPV target DNA. By the way, after an interval of 5 days, 36 days and 78 days, only about 1.14%, 2.79% and 3.30% degeneration in the fluorescence response were demonstrated respectively (Fig.S9), which indicated the excellent stability of Fe3O4@Au@PEI@HPP NPs.


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3.6 DNA sensing on Fe3O4@Au@PEI@HPP@Probe NPs in buffer or in whole serum Optimization

of

the

parameters

of

DNA

sensing

on

Fe3O4@Au@PEI@HPP@Probe NPs was firstly investigated in detail. As shown in Fig.S10, 15 min and 5 min were respectively selected as the optimal reaction time for the interaction between probe and target and the optimal hybridization time between fluorescent dyes and double DNA. Then, the analytical performance of Fe3O4@Au@PEI@HPP@HPV18 NPs for detecting HPV18-CS was investigated in 100% serum, containing a series of HPV18-CS concentrations using fluorescent method. Encouragingly, as shown in Fig.4B, with an increase in the concentration of HPV18-CS (from 5 nM to 1 μM), the fluorescence intensity of all samples at 450 nm increased gradually and the corresponding regression equation was F=-16.90+91.10×log[CHPV182 CS] with the correlation coefficient (R ) of 0.9966 (Fig.4C, 4D). Interestingly, for HPV16,

a good linear correlation (F=-3.511+83.69×log[CHPV16-CS], R2=0.9984, Fig.S11C, S10D)was also exhibited. The corresponding limit of detection (LOD) for both samples were 1.78 nM and 1.23 nM, respectively. Those results were basically consistent with that in buffer (HPV18: F=-16.38+86.63×log[CHPV18-CS], R2=0.9942, Fig.4A, 4B; HPV16: F=-25.38+89.21×log [CHPV16-CS], R2=0.9972, Fig.S11A, S10B). All of data indicated that application of Fe3O4@Au@PEI@HPP NPs on disease marker assays has its own unique advantages (Table S2). This should be emphasized that for experiments performed in whole serum, the interference of Fe3O4@Au@PEI@HPP@probe NPs (control 5) and Fe3O4@Au@PEI@HPP@probe NPs+Hoechst 33258 (control 6) is very small, so control 6 is selected as the background value, namely F0 (Fig.S7B). 3.7Analytical performance of Fe3O4@Au@PEI@HPP@Probe NPs



In order to evaluate the practical application of Fe3O4@Au@PEI@HPP@Probe NPs, the standard addition method was performed to detect the concentration of HPV18/16 target DNA (40 nM or 400 nM) in 100% serum samples. The results are listed in Table 1, the recoveries for the detection of HPV18/16 target DNA in 100% serum ranged from 97.46% to 102.46% with the relative standard deviation (RSD) from 2.53% to 2.78%, which implied that Fe3O4@Au@PEI@HPP@Probe NPs have great practical application prospect.

4 Conclusions In this work, a novel gold magnetic nanoparticle based on PEI and HPP was synthesized by a simple route. Moreover, it demonstrated an excellent dispersibility, stabilization, biocompatibility, efficient target recognition, especially antifouling ability. Therefore, based on this material, a strategy for rapid, facile and sensitive identification of HPV genotype 18, 16 in buffer, even in whole serum was developed. More interestingly, in 100% serum, probe based on this novel nanomaterial showed a very good selectivity with complementary DNA and can be used in a wide range of detection area (5 nM~1 μM) under the optimum conditions. In addition, the sensitive detection of limits of HPV18 or HPV16 target is 1.78 nM or 1.23 nM, respectively. All basically match what is shown in buffer. In a word, the most prominent innovation of this report is that the goal of using magnetic beads as probes to directly and rapidly diagnose diseases in whole serum has been achieved, suggesting the great economic value of this material.

ASSOCIATED CONTENT Supporting Information


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The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details and additional figures.(PDF) AUTHOR INFORMATION Corresponding Authors E-mails: [email protected] and [email protected]; Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the science and technology project and achievement transformation plan of modern agricultural of Xinjiang Corps (2016AC010), The National Science and Technology Major Project (2017YFD0500304), Science and technology branch project of Xinjiang autonomous region, science and technology project to support Xinjiang autonomous region (2018E02021) and The Applied Basic Research Program of Qingdao (17-1-1-65-jch).



Scheme

1.

Schematic

Illustration

of

the

fabrication

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procedures

of

Fe3O4@Au@PEI@HPP NPs and the strategy of HPV genotype detection in buffer or in 100% serum based on Fe3O4@Au@PEI@HPP NPs


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Fig.1 Hysteresis loops of Fe3O4@Au@PEI@HPP NPs (A); Photograph of Fe3O4@Au@PEI@HPP NPs without magnetic field (B, a), with magnetic field(B, b); HR-TEM

image

of

Fe3O4@Au@PEI@HPP

NPs

(C);

TEM

image

of

Fe3O4@Au@PEI@HPP NPs (D); Size distribution of Fe3O4@Au@PEI NPs using DLS (E); SEM image of Fe3O4@Au@PEINPs (F); SEM image of Fe3O4@Au@PEI@HPP NPs

(G).

FTIR

spectra

of

Fe3O4@Au@PEI

Fe3O4@Au@PEI@HPP NPs (H, curve b).


NPs

(H,

curve

a)

and


Fig.2 Antifouling property of Fe3O4@Au NPs, Fe3O4@Au@PEI NPs and Fe3O4@Au@PEI@HPP NPs evaluated by DPV method of electrochemistry in different media (1 μM DNA, 10 mg mL-1 BSA, and 100% serum).


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Fig.3 Selectivity evaluation of probe based on Fe3O4@Au@PEI@HPP NPs to HPV genotype 18



Fig.4 Fluorescence spectra on addition of different concentrations (5 nM~1 μM) of HPV18-CS in buffer (A) or in 100% serum (C). The linear relation between fluorescence intensity (F-F0) and the concentrations of HPV18-CS in buffer (B) or in 100% serum (D).


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Table1: Results of detection of HPV genotype 18, 16 in 100% serum Samples

Added

Found (nM)

(nM)

Recovery (%)

RSD (%, n=3)

1-HPV16

40.0

40.49

101.24

2.78

2-HPV16

400.0

408.4

102.1

2.75

1-HPV18

40.0

40.98

102.46

2.53

400.0

388.6

97.16

2.46

2-HPV18



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