Mechanistic Studies of Enhanced PCR Using PEGylated PEI

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Mechanistic Studies of the Enhanced PCR Using PEGylated PEI-Entrapped Gold Nanoparticles Aijun Li, Benqing Zhou, Carla S. Alves, Bei Xu, Rui Guo, Xiangyang Shi, and Xueyan Cao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09310 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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ACS Applied Materials & Interfaces

Mechanistic Studies of the Enhanced PCR Using PEGylated PEI-Entrapped Gold Nanoparticles Aijun Li a, Benqing Zhou a, Carla S. Alves b, Bei Xu a, Rui Guo a, Xiangyang Shi *, a, b, and Xueyan Cao *, a

a

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai

201620, People’s Republic of China b

CQM-Centro de Quimica da Madeira, Universidade da Madeira, Campus da Penteada, 9020-105

Funchal, Portugal

*To whom correspondence should be addressed. E-mail: [email protected], Tel: +86-21-67792656; E-mail: [email protected], Tel: +86 21 67792750, Fax: +86 21 67792306-804.

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ACS Paragon Plus Environment


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ABSTRACT: The polymerase chain reaction (PCR) is considered as an excellent technique and is widely used in both molecular biology research and various clinical applications. However, the presence of byproducts and low output are limitations generally associated with this technique. Recently, the use of nanoparticles (NPs) has been shown to be very effective at enhancing PCR. Although mechanisms underlying this process have been suggested, most of them are mainly based on PCR results under certain situations without abundant systematic experimental strategy. In order to overcome these challenges, we synthesized a series of polyethylene glycol (PEG)-modified polyethyleneimine (PEI)-entrapped gold nanoparticles (PEG-Au PENPs), each having different gold loading contents. The role of the synthesized NPs in improving the PCR technique was then systematically evaluated using the error-prone two-round PCR and GC-rich PCR (74% GC content). Our results suggest a possible mechanism of PCR enhancement. In the error-prone two-round PCR system, the improvement of the specificity and efficiency of the technique using the PEG-Au PENPs mainly depends on surface charge-mediated electrostatic interaction. In the GC-rich PCR system, thermal conduction may be the dominant factor. These important findings offer a breakthrough in understanding the mechanisms involved in improving PCR amplification, as well as the application of nanomaterials in different fields, particularly in biology and medicine.

KEYWORDS: polymerase chain reaction; PEG-Au PENPs; thermal conductivity; electrostatic interaction; improvement

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INTRODUCTION The polymerase chain reaction (PCR) is considered one of the most meaningful techniques in the postgenomics era and has extensive applications in many fields.1-3 However, PCR has faced many technical challenges including limitations in DNA availability in samples, as well as low efficiency and specificity in amplification.4 Many important genes contain high GC regions. However, owing to their high melting temperature and secondary structures,5 DNA fragments are difficult to amplify. While traditional methods and commercially available PCR kits play a role in improving this technique, they do not always work and are often unpredictable.6 Over the last 20 years, the use of nanomaterials have drawn great attention due to their unique physical and chemical properties including excellent thermal conductivity, high surface to volume ratios and surface charge density.7 To date, various nanomaterials such as gold nanoparticles (Au NPs),8 carbon nanotubes (CNTs),9-10 graphene oxide (GO),11 graphene nanoflakes (GNFs),12 poly (amido-amine) (PAMAM) dendrimers 13 and quantum dots (QDs)14-15 have been utilized to improve DNA amplification technologies. Although several potential mechanisms for the effects of nanomaterials have been proposed, most of them are mainly conjectural based on PCR products under certain situations without abundant systematic experimental strategy.8, 11, 14 Considering the complexity of the interactions between nanoparticles (NPs) and PCR components, the detailed mechanisms are likely to be very complex. In our previous work, we found that the enhancing effects of NPs maybe related to their electrostatic interaction with the PCR components, as well as their heat conductivity properties.16-17 In order to further investigate the mechanisms involved in this process, the development of various NPs with different surface characteristics and thermal conductivity would be necessary. Branched polyethyleneimine (PEI) is a highly branched polymer made up of repeat units of the amine group, including primary, secondary and tertiary amino groups. It exhibits good solubility, low solution viscosity and a high degree of functionality.18 As a result of its unique structure, branched PEI can be used as a stabilizer to entrap inorganic NPs. Au NPs have been subject to much 3



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attention on account of their unique physicochemical properties and simplicity in preparation and 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

surface modification. In our previous work, polyethylene glycol (PEG)-modified PEI was reported as a model for the synthesis of Au NPs and the developed PEG-modified PEI NPs were used to prepare PEGylated PEI-entrapped Au NPs (PEG-Au PENPs) using different Au atom/PEI molar ratios.19 To explore a possible PCR optimization mechanism, a similar approach was used in the present study which allowed us to coordinate the NP composition, morphology, and surface functionalization of the same particle system for different surface charge characteristics and thermal conductivities. In this study, a series of PEG-Au PENPs having different Au/PEI molar ratios were synthesized using branched PEI as a template, similar to our previously published protocol.19 The prepared NPs were utilized as additives for improvement of the two-round PCR system and the GC-rich PCR system (74% GC content). The influence of the different PEG-Au PENPs on the efficiency and specificity of the PCR system under investigation was determined by means of a systematic and comprehensive approach. Zeta potential, size and thermal conductivity of the PEG-Au PENPs were also measured. The associated mechanisms were also discussed for each respective PCR system. We believe that these results will provide valuable insights into the application of PEG-Au PENPs and other nanomaterials in the fields of biology and medicine.

EXPERIMENTAL SECTION Materials. Amine-terminated pristine branched PEI (molecular weight = 25,000) was purchased from Aldrich (St. Louis, MO). PEG monomethyl ether with a carboxyl end group (mPEG-COOH, Mw = 2,000) was from Shanghai Yanyi Biotechnology Corporation (Shanghai, China).

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride

(EDC)

and

N-hydroxysuccinimide (NHS) were from J&K Chemical Ltd. (Shanghai, China). HAuCl4·4H2O and acetic anhydride were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Synthesis and Characterization of PEG-Au PENPs. mPEG-COOH (30.0 mg, 0.015 mmol, 4


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3.0 mL in water) was activated by EDC/NHS (10 molar equivalents of mPEG-COOH, 28.75 mg 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

EDC and 17.25 mg NHS) under magnetic stirring for 3 h at room temperature. Then, mPEG-COOH was dropped into a PEI solution (12.5 mg, 0.5 µmol, 5.0 mL in water) under magnetic stirring at room temperature and the reaction mixture was stirred for 72 h. The reaction mixture solution was dialyzed against phosphate buffered saline and water using a dialysis membrane with an MWCO of 14,000 for 72 h, followed by a freeze drying process to obtain the PEI·NH2-mPEG product. The PEG-Au PENPs with gold salt (HAuCl4)/branched PEI molar ratio at 100 : 1, 200 : 1, and 300 : 1 were synthesized and characterized according to our previous work.19 NaBH4 reduction chemistry was applied to prepare various kinds of PEG-Au PENPs. 1H NMR spectra were collected on a Bruker DRX 400 nuclear magnetic resonance spectrometer. UV-vis spectroscopy was carried out using a Lambda 25 UV-vis spectrophotometer (Perkin Elmer, Boston, MA). The synthesized PEG-Au PENPs are quite stable in water, PBS, and cell culture medium and don’t precipitate for at least 3 months.19 To avoid introducing any other components to PCR system, PEG-Au PENPs were dispersed in sterilized double-distilled water before all of the experiments. A JEOL 2010F analytical electron microscope (JEOL, Tokyo, Japan) was used to characterize the size and morphology of the samples at an operating voltage of 200 kV via transmission electron microscopy (TEM) imaging. TEM samples were available by dropping an aqueous particle suspension (1 mg/mL) onto a carbon-coated copper grid and the aqueous suspension was air dried before measurements. Zeta potential and dynamic light scattering (DLS) were tested using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, UK) coupled with a standard laser with a wavelength of 633 nm. Two-Round PCR Test System. The established error-prone two-round PCR system was similar to the model described in our previous work.13 A 283-bp DNA fragment was amplified from λDNA (Takara Bio Inc.) using one pair of primers (Shanghai Sangon Biological Engineering and Technology and Service Co. Ltd.). Primer 1: 5’-GGCTTCGGTCCCTTCTGT-3’, Primer 2: 5’-CACCACCTGTTCAAACTCTGC-3’. PCR reagents were mixed in a final volume of 25 µL according to the following conditions: 10×reaction buffer, 0.2 µM primers, 0.25 mM each dNTP, 5



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and 0.025 U/µL rTaq DNA polymerase (Takara Bio Inc.). The PCR procedure was: 2 minutes at 94 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

o

C for pre-denaturation, followed by 35 cycles of: 30 s at 94 oC, 30 s at 57 oC, 30 s at 72 oC. The

final extension was performed at 72 oC for 5 minutes. Amplifications were carried out in a S1000TM Thermal Cycler (Bio-Rad Inc.). GC-Rich

PCR

Test

System.

In

5’-ACAGAATTCGCCCCGGCCTGGTACAC-3’

GC-rich and

PCR

system, Primer

Primer

3: 4:

5’-TAAGCTTGGCACGGCTGTCCAAGGA-3’ were designed to amplify a 264-bp GC-rich fragment (74% GC content) from 200 ng human genomic DNA (Chembio-engine. Biomart.). The amplification mixture was similar to those mentioned above except that 0.15 µM primers and 0.05 U/µL rTaq DNA polymerase were added to a final volume of 25 µl. The PCR protocol was: 5 minutes at 94 oC for pre-denaturation, followed by 35 cycles of: 1 min at 94 oC, 2 min at 64.2 oC, 3 min at 72 oC. Then the cycling was terminated after incubation at 72 oC for 5 min. Evaluation Methods of PCR. The PCR products were analyzed by the agarose gel electorphoresis (1.2 W/V %) stained with ethidium bromide, and visualized and photographed on a FR-980A gel image analysis system (Shanghai Furi Science&Technology Co., Shanghai, China). DL2000 Marker (Takara Bio. Inc.) was employed to mark the size of DNA. The capability of the tested additives to increase the efficiency of amplification was defined as a ratio of the densitometric value of the target DNA band determined after PCR to 500-bp band of DL2000 Marker. The specificity of amplification was figured out as a ratio of the densitometric value of the specific band and that of all bands, including undesired non-specific bands, amplified by PCR. The specificity in the absence of non-specific bands was defined as the maximal value that equals to 1.0. The concentration of each additive that made PCR produce the maximal specificity and the brightest target band was identified to be the optimum concentration. Each reaction was performed three times, and all the PCR products were analyzed by sequencing after purification in order to determine the fidelity of PCR. Thermal Conductivity Measurements. Thermal conductivity of the PEG-Au PENPs was 6


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detected by a KD2 Pro portable thermal conductivity meter (Decagon Devices, U.S.A). The transient 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

line source (TLS) uses a kind of sensor to test materials. Every measurement cycle includes 30 s of each balance, heating and cooling time. The temperature measurement value was displayed every other 1 s. Equipment operating environment temperature is from 20 oC to 60 oC.

Results and Discussion Preparation and Characterization of PEG-Au PENPs. Three kinds of PEG-Au PENPs with the gold atom/PEI molar ratio at 100: 1, 200: 1, and 300: 1 (denoted as {(Au0)100-PEI-mPEG24} NPs, {(Au0)200-PEI-mPEG24} NPs and {(Au0)300-PEI-mPEG24} NPs, respectively) were successfully synthesized and characterized. The synthesis processes of formed PEG-Au PENPs with different Au atom/PEI molar ratio were illustrated in the above and further visualized in Scheme 1. As shown in Figure 1, it is apparent that the peaks at 2.4-3.5 ppm can be assigned to the -CH2proton signals of PEI, while the peaks at 3.5-3.8 ppm are associated with the -CH2- protons of PEG.20-22 According to the comparison of the NMR integration, we figured out the number of mPEG linked with each PEI is about 24. This number is approximately similar to the original molar feeding ratio (mPEG-COOH/PEI ≈ 30:1). The hydrodynamic size and zeta-potential measurements of materials were characterized to examine the surface potential and particle diameter. The results displayed the surface potential of particle increases with the growing of the Au atom/PEI molar ratio (Table 1). And the diameters of materials also measured by DLS spectroscopy, following the order of {(Au0)100-PEI-mPEG24} NPs (211.4 nm) > {(Au0)200-PEI-mPEG24} NPs (188.3 nm) > {(Au0)300-PEI-mPEG24} NPs (156.3 nm). It is clear that all PEG-Au PENPs display an absorption peak at 520 nm (Figure 2) with a higher peak intensity at a higher Au atom/PEI molar ratio by UV-vis spectroscopy, which verifies the successful formation of PEG-Au PENPs.23-24 Then we used TEM to test the size and morphology of the Au core NPs in prepared {(Au0)n-PEI-mPEG} NPs (Figure 3). The images illustrated that the diameter of PEG-Au PENPs at a 7



given Au atom/PEI molar ratio is small and consistent. The size of the Au core NPs can be varied 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

from 4.5 to 6.9 nm and gradually increase with the growing of the Au atom/PEI molar ratio, in according with the results of hydrodynamic size. Effects of PEG-Au PENPs on Error-Prone PCR Amplification. To test whether or not PEG-Au PENPs had the ability to improve performances of PCR amplification, we first chose an error-prone two-round PCR system as a template to examine the effects of these NPs. The two-round PCR system was similar to the model described in our previous work.16 On the ground of the same primes used in two rounds of PCR, it usually failed to amply any meaningful products in the second round. Even with optimization of the concentration of Mg2+ or annealing temperature, these “error-prone” non-specific amplification couldn’t be eliminated.25 Based on our experiments, varying the concentration of magnesium ions couldn’t obtain a single and bright target band (Figure S1). So it may be an appropriate model to test the effects of NPs on the PCR performance. The PEG-Au PENPs were detected in this PCR system and PEI alone was also tested as a control. All of the materials were used at varying concentrations in the PCR mixture. As shown in Figure 4, it is clear that all the PEG-Au PENPs can strengthen the efficiency and specificity of the two-round PCR amplification. The target band was brighter, while the non-specific band decreased with the increasing concentration of the additives. However, when the concentrations exceeded the optimum concentration, all the amplification was totally inhibited, as similar to other conventional PCR additives.16 In addition, we found that when the concentration of PEG-Au PENPs increased to a certain degree or beyond their corresponding optimum concentration, the non-specific products approximately came back to the original state. This phenomenon is quite different from that of control PEI (Figure 4a) and our previous reports,13 in which the target band just gradually decreased until disappeared. Despite the exact reason is unknown, some reports investigated the influence of the mPEG on PCR components. Xun et al.26 used mPEG to conjugate the QD surface and found that this modification enables QDs to endure 40 thermal cycles in the presence of PCR components. We thought that the PEG modified in the PEG-Au PENPs was reported to resist protein adsorption,27-29 probably leading to weak interaction between PEG-Au PENPs and DNA polymerase and 8


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subsequently loss of the effect on improvement when the concentrations of additives were slightly 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

over the corresponding optimum concentrations. Besides, when the concentrations were much higher than the optimum concentrations, the targets amplification was inhibited like other nanomaterials. Based on semi-quantitative analysis, we calculated the PCR efficiency and specificity in presence of each additive according to ImageJ software (Table 1). The optimum concentrations of PEI, {(Au0)100-PEI-mPEG24} NPs, {(Au0)200-PEI-mPEG24} NPs and {(Au0)300-PEI-mPEG24} NPs are 0.47, 0.38, 0.34, 0.28 mg/L, respectively. As we known, branched PEI are soft, flexible and prone to take shape a flat balloon which goes against reacting with other material.16 Due to the gold loading content, PEG-Au PENPs could form 3D spherical morphology, providing more binding sites to other materials, such as PCR components.30 Accordingly, it is not surprising to see that the optimum concentration of PEG-Au PENPs decreased with the increasing Au atom/PEI molar ratio. To further prove the proposed mechanism, acetylated {(Au0)200-PEI-mPEG24} NPs, namely {(Au0)200-PEI-NHAc-mPEG24} NPs, was employed to optimize the same PCR system (Figure 5). For the decreased number of amine terminal groups caused by the acetylation of the surface amino groups, so the zeta potential of {(Au0)200-PEI-NHAc-mPEG24} NPs reduced, which weakens interaction with PCR components.16 Although {(Au)200-PEI-NHAc-mPEG24} NPs also had the effect of improving the efficiency and specificity of PCR, the optimum concentration (60 mg/L) was about 176 times higher than that of the {(Au0)200-PEI-mPEG24} NPs (0.34 mg/L). With the surface potential of additives having an order of {(Au0)200-PEI-NHAc-mPEG24} NPs (+6.34 mV) < PEI (+24.07 mV) < {(Au0)100-PEI-mPEG24} NPs (+28.93 mV) < {(Au0)200-PEI-mPEG24} NPs (+33.46 mV) < {(Au0)300-PEI-mPEG24} NPs (+34.23 mV), the optimum concentration follows the order of {(Au0)200-PEI-NHAc-mPEG24} NPs > PEI > {(Au0)100-PEI-mPEG24} NPs > {(Au0)200-PEI-mPEG24} NPs > {(Au0)300-PEI-mPEG24} NPs. The above results suggested that the surface charge-mediated electrostatic interaction between the positively charged NPs and negatively charged PCR components presumably played an important role in optimizing the error-prone PCR amplification. Effects of PEG-Au PENPs on GC-Rich PCR Amplification. In order to further verify the ability of PEG-Au PENPs on optimizing PCR products, we also established another template with 9



high GC content. Apolipoprotein E (ApoE) gene is situated on chromosome 19, known as a high GC 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

content segment. It is said alleles of ApoE gene have some relation with some diseases, and have been produced as a result of single nucleotide polymorphism (SNP) mutation.31 Based on the published reports, traditional methods did not illustrate prominent and reproducible results of ApoE gene.32 Multiple smear and non-specific bands were generated even though the PCR reaction needed to take up to four hours. Considering the effect of Mg2+ on yield and specificity, we first performed the amplification of ApoE gene with different concentrations of Mg2+ and found no improvement of target product (Figure S2). After adding the test materials to PCR mixture, the target fragments appeared with the non-specific bands fading (Figure 6). The optimum concentrations of {(Au0)100-PEI-mPEG24} NPs, {(Au0)200-PEI-mPEG24} NPs and {(Au0)300-PEI-mPEG24} NPs were estimated to be 0.80, 0.60, 0.56 mg/L, respectively. Consistent with our previous results, PCR was totally inhibited when the added concentration exceed a certain value. It is essential to note that the optimum concentration of PEI (12 mg/L) is 15 times higher than that of PEI entrapped Au NPs in GC-rich PCR system, while almost the same in two-round PCR system. The obvious distinction of the optimum concentrations of PEI in two PCR systems probably revealed the existence of two different mechanisms. In order to further explore whether the improvement is related to the Au NP-mediated thermal conductivity, GC-rich PCR reactions with a reduced time of cycles were also conducted. We decreased the duration of each step within a three-step cycle from 1 min to 45 s at 94 oC, 2 min to 90 s at 64.2 oC, and 3 min to 60 s at 72 oC, which resulted in a final PCR reaction time of only 2h. The electrophoresis gel images showed that the targets can be obtained with the assistance of PEG-Au PENPs, even the denaturation time was reduced to only 25 s, while no products could be amplified in the positive control system with PEI added (Figure 7). It is interesting that the optimum concentrations of {(Au0)100-PEI-mPEG24} NPs (0.32 mg/L), {(Au0)200-PEI-mPEG24} NPs (0.24 mg/L) and {(Au0)300-PEI-mPEG24} NPs (0.20 mg/L) are less than those in the original GC-rich PCR system, similar to those in two-round PCR system. It is presumably due to the emergence of non-specific bands in original GC-rich PCR. More PEG-Au PENPs were needed to ensure the 10


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correct amplification and inhibit the mispairing between primers with DNA templates when the 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

duration of PCR cycle was longer. Enhancement Mechanism. Since the introduction of nanoPCR in 2005, a wide range of NPs have been successfully used to enhance the specificity and efficiency of PCR. For example, Park et al.33 showed that the dopamine-assisted synthesis of carbon-coated silica could significantly enhance the performance of PCR. Xun et al.26 synthesized a QD590–mPEG conjugate which was found to be a stable fluorescent sensor in the PCR reaction capable of enhancing thermal cycling durability and PCR compatibility. And the ammonium salt of oleic acid coated magnetite (Fe3O4) NPs effectively improved PCR amplification efficiency relative to the NPs of Au and Ag.34 Several possible mechanisms for the effects of these nanomaterials have been proposed, such as (1) mimic the function of single-stranded DNA (ssDNA)-binding protein (SSB) to selectively bind ssDNA rather than double-stranded DNA (dsDNA). Li et al.8 postulated that the PCR enhancement observed for their nanoPCR system may be a result of the Au NPs mimicking the function of SSB. Here, the NPs may selectively bind ssDNA and not dsDNA, largely minimizing the mispairing between primers and DNA during the PCR process. (2) Absorption of DNA polymerases to regulate the concentration of enzyme in PCR solution. Vu et al.35 and Mi et al.36 demonstrated that Au NPs adsorb onto DNA polymerases and dynamically interact with it to reduce non-specific fragment in PCR solution. Lou et al.37 further demonstrated that the Au NPs effects on PCR enhancement were not only caused by polymerase adsorption but also by primer and product adsorption. (3) Catalytic property of NPs. Cui et al.38 investigated via quantitative PCR product measurements and various other techniques the effects of SWCNTs on PCR were caused by catalytic property of SWCNTs, because similar results have been obtained in PCR reactions in the presence and in the absence of Mg2+ ions in serving as electron donors/receptors. While the above data provide insights into the observed phenomena, the exact mechanisms of PCR enhancement with nanomaterials are still unknown and need more systematic investigation. In our previous work, PEI-modified CNTs with different surface charge polarities16 were used as enhancers of PCR specificity and efficiency. It was found that for positively charged CNT/PEI, 11



electrostatic interaction played an important role in concentrating the PCR components locally on 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

the NP backbones thereby increasing the probability of the dynamic contact between each element. We have also demonstrated that positively charged dendrimers have better optimization effects relative to neutral or negatively charged dendrimers17 and with Au NPs entrapped, the optimum concentration significantly decreased.30 In accordance with our previous work, Yuan et al.39 and Yüce et al.40 also showed that the reason of enhanced PCR amplification could be that DNA templates may bind onto positively charged NPs by electrostatic interaction resulting in enhanced PCR amplification. For negatively charged NPs on the other hand, an inhibitory action on PCR amplification was detected attributable to the electrostatic repulsion between the negative charges of the different elements. Branched PEI used in this study, which has some characteristics in common with dendrimers (PAMAM), is a polycationic synthetic polymer that is soft, flexible and has a flat balloon shape.16 As such, we used it as a template to synthesize PEG-Au PENPs for further exploring the mechanisms associated with PCR enhancement. In our study, it is clear that the zeta potential of the PEG-Au PENPs gradually increased for the more reserved 3D spherical morphology with an increase in Au loading content (Table 1), suggesting more terminal amino exposure.30 It is likely that the maintaining of 3D spherical morphology of the PEG-Au PENPs provides more reaction opportunity between NPs and PCR components, resulting in a decrease in the optimum concentrations used in the error-prone two-round PCR system. The PCR enhancement efficacy however was weakened after surface acetylation of the PEG-Au PENPs, due to the reduction in the number of amine terminal groups. These results suggest that the electrostatic interaction between the positively charged NPs and negatively charged PCR components has a great influence on improving PCR specificity and efficiency. For a highly efficient amplification reaction to occur, the attachment of the PCR components onto the positively charged NPs may facilitate primer pairing and thus DNA polymerase binding, as well as increase the local concentrations of the PCR components. Similar views have been reported by others.16, 40 The thermal conductivity of the NPs is also believed to be a crucial factor in nanoPCR. Many 12


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studies have reported that the excellent thermal conductivity properties of Au NPs may be the main 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

reason for improving PCR performance. Li et al.41 reported that the excellent thermal conductivity of the Au NPs should be the essential factor in improving the PCR efficiency. When cutting down PCR time, the efficacy of PCR with Au NPs was increased compared with the PCR components without Au NPs. Besides, Au NPs can improve the PCR performance in the quicker thermal cycler (higher heating/cooling rate). Yang et al.4 investigated Au NPs could improve PCR performance of several high GC content PCR systems, probably because Au NPs could facilitate double-stranded DNA dissociation. Amplification of templates with a high GC content using PCR is usually hampered due to the formation of secondary structures like hairpins, higher melting temperatures and mispairing32, 42 compared to non-GC-rich targets. The addition of nanoparticles in PCR system results in enhanced thermal conductivity effect, which allows the DNA, Taq DNA polymerase and other reagents to interact more efficiently. Khaliq et al.12 showed that GNFs can improve the thermal conductivity of base fluids to enhance PCR efficiency. They proposed a hypothesis for PCR enhancement that GNFs can help better heat dissipation in PCR, which leads to more rapid DNA denaturation during the denaturation stage; then the van der Waals forces among NPs may contribute to binding with PCR reagents for enhanced interaction during the second stage; at last, the third stage might have been improved again because of good dissipation of heat, finally resulting in the improvement of PCR products. In addition, Cui et al.43-44 also suggested the similar NPs enhancement mechanisms which the thermal entropy of the NPs is in favor of dynamic contact among PCR elements, and reduces the mispairing between primers and templates at the each step during PCR process. Thus, a high GC-content PCR system should be an ideal template to study heat transfer effect of nanomaterials. To ensure the specificity and yield of GC-rich DNA amplification without the assistance of any additives, the duration of template denaturation is usually extended to maintain the stability of ssDNA, and the annealing temperature is increased to reduce the mispairing between primers and templates. However, it always causes a failure of target DNA amplification. In our study, we chose a GC-rich PCR system (a 264-bp ApoE gene, 74% GC content) to explore the effect of thermal 13



conductivity on PCR improvement. The results showed that PEG-Au PENPs had much desired 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

effect compared with PEI alone. Under the optimal concentrations of PEG-Au PENPs, the target fragment could be successfully obtained. In contrast, no products were amplified without additives following the common GC-rich PCR protocol. Besides, the optimum concentration of PEI is about 15 times higher than that of PEG-Au PENPs, which quite differ with the concentrations used in two-round PCR system. When we shortened the duration of PCR cycles to non-GC-rich PCR protocol, only PEG-Au PENPs were capable of conducting the GC-rich DNA amplification. In this case, the electrostatic interaction mechanism with respect to PEI additives cannot explain this phenomenon.16 Thus, the heat conduction mediated by PEG-Au PENPs seems a key role in enhancing GC-rich PCR amplification. It is well known that the hydrogen bonds in high GC content DNA are hard to fracture at denaturation temperature. Due to better dispersion of heat of PCR solution at the presence of PEG-Au PENPs, the GC-rich sequence is supposed to denature more rapidly. This good dissipation of heat may be a result of the collision between the PCR components and the PEG-Au PENPs. To further confirm this view, we further tested the thermal transfer efficiency of our additives (Figure 8). Here, the solution that contained PEG-Au PENPs took much less time to reach a certain temperature relative to the PEI-containing solution. The thermal conductivity coefficient also stated this point (Table 2), following the order of {(Au0)300-PEI-mPEG24} NPs (1.296 Wm·K) > {(Au0)200-PEI-mPEG24} NPs (1.105 Wm·K) > {(Au0)100-PEI-mPEG24} NPs (0.753 Wm·K) > PEI (0.552 Wm·K). When the duration of the PCR amplification cycle is long enough, the target band may also be obtained using a higher optimum concentration of PEI, despite its lower thermal conductivity. While the times of the three steps were all reduced, PEI had no effect on the improvement of PCR. These results imply that the high GC content PCR system with the added aqueous suspension of PEG-Au PENPs would have an excellent thermal conductivity, leading to a rapid thermal equilibrium and convection heat transfer. Thus, it would be useful for GC-rich PCR to improve the specific annealing of primers with templates, enhance the yield of target DNA and decrease the non-specific formation of fragments. From these results we thought the enhancement of 14


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GC-rich PCR by PEG-Au PENPs could be attributed to Au NPs’ ability to increase thermal 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

conductivity rather than facilitate the dissociation of dsDNA45-46 and influence the interaction with the DNA polymerase.4, 36 It turns out that the improvement of the specificity and efficiency of PCR mainly depends on the surface charge-mediated electrostatic interaction in error-prone two-round PCR system, while thermal conduction might be the dominant factor in GC-rich PCR system.

CONCLUSION In summary, the most important result in this study is the identification of PEG-Au PENPs that have the potential capacity to enhance the specificity and efficiency in both two-round PCR and GC-rich PCR. In accordance with our previous literature, the optimum concentrations of PEG-Au PENPs decreased depending on the increase of the molar ratio between Au atom and PEI. The enhancement mechanism behind nanomaterials is related not only to the effects of electrostatic effect, but also the effect of the thermal conduction, depending on the type of PCR system. We confirmed PEG-Au PENPs as a general additive in PCR amplification, which is significant in the field of molecular biology and clinical medicine.

ASSOCIATED CONTENT Supporting Information The effect of the magnesium ions on two-round PCR, original GC-rich PCR and modified GC-rich PCR supplied as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: +86-21-67792656. * E-mail: [email protected]. Tel: +86-21-67792750. 15



Notes 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

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research is financially supported by the National Natural Science Foundation of China (31400816), and the Fundamental Research Funds for the Central Universities (X. C.). X. C. thanks the Shanghai Pujiang Program (13PJD026) and the Key Program of Science and Technology Commission of Shanghai Municipality (13NM1401700) for financial support. A. L. thanks the Innovation Funds of Donghua University Master Dissertation of Excellence (EG2016007).

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21



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Table 1. The physicochemical properties and optimum concentrations of the additives in the error-prone two-round PCR.

Additives

Zeta Potential (mV)

Optimum Concentration (mg/L)

*Maximal efficiency

*Maximal specificity

PEI

24.07 ± 1.45

0.47

1.5

1

{(Au0)100-PEI-mPEG24} NPs

28.93 ± 0.85

0.38

2.2

1


33.46 ± 1.28

0.34

3.6

1


34.23 ± 1.09

0.28

1.9

1

{(Au0)200-PEI.NHAc-mPEG24} NPs

6.34 ± 1.13

60

1.4

1

*depend on the performance of each additive with optimum concentration.

Table 2. The thermal conductivity of the additives (Mean ± S.D., n = 3).

Additives

PEI

{(Au0)100-PEImPEG24} NPs

{(Au0)200-PEI-m PEG24} NPs

{(Au0)300-PEI-m PEG24} NPs

Thermal conductivity coefficient(W/(m·K))

0.552 ± 0.011

0.753 ± 0.013

1.105 ± 0.009

1.296 ± 0.016

22


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Figure captions: 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

Scheme 1. Schematic illustration of the synthesis of PEGylated Au PENPs. Figure 1. 1H NMR spectra of PEI-mPEG. Figure 2. UV-vis spectra of the {(Au0)n-PEI-mPEG} NPs (n = 100, 200, and 300, respectively). Insets show the respective photographs of the suspension of the PEG-Au PENPs (0.1 mg/mL). Figure 3. TEM images and size distribution histograms of the {(Au0)n-PEI-mPEG} NPs (n = 100 (a), 200 (b), and 300 (c), respectively). Figure 4. The effect of the PEI and PEG-Au PENPs on two-round PCR. In each image, lane M is for DNA marker, and the last lane is the negative control. (a) PEI was added into the PCR mixture, and for lane 1 to lane 6, its final concentration was 0, 0.40, 0.45, 0.47, 0.49, 0.52 mg/L, respectively. (b) {(Au0)100-PEI-mPEG24} NPs was added into the PCR mixture, and for lane 1 to 6, its final concentration was0, 0.30, 0.36, 0.38, 0.42, 0.46 mg/L, respectively. (c) {(Au0)200-PEI-mPEG24} NPs was added into the PCR mixture, and for lane 1 to 5, its final concentration was 0, 0.32, 0.34, 0.39, 0.43 mg/L, respectively. (d) {(Au0)300-PEI-mPEG24} NPs was added into the PCR mixture, for lane 1 to 6, its final concentration was 0, 0.20, 0.24, 0.28, 0.34, 0.38 mg/L, respectively. Figure 5. The effect of the {(Au0)200-PEI.NHAc-mPEG24} NPs on two-round PCR. Lane M is for marker. For lane 1 to lane 6, its final concentration was 0, 20.0, 40.0, 60.0, 70.0, 80.0 mg/L, respectively, lane 7 is the negative control. Figure 6. The effect of the PEI and PEG-Au PENPs on GC-rich PCR. In each image, lane M is for marker, and the last lane is the negative control. (a) PEI was added into the PCR mixture, and for lane 1 to lane 5, its final concentration was 0, 10.0, 12.0, 14.0, 16.0 mg/L, respectively. (b) {(Au0)100-PEI-mPEG24} NPs was added into the PCR mixture, and for lane 1 to 6, its final concentration was 0, 0.60, 0.80, 1.00, 1.20, 1.40 mg/L, respectively. (c) {(Au0)200-PEI-mPEG24} NPs was added into the PCR mixture, and for lane 1 to 6, its final concentration was 0, 0.50, 0.56, 0.60, 0.80, 1.00 mg/L, respectively. (d) {(Au0)300-PEI-mPEG24} NPs was added into the PCR mixture, for lane 1 to 5, its final concentration was 0, 0.40, 0.56, 0.60, 0.80 mg/L, respectively. Figure 7. The effect of the PEI and PEG-Au PENPs on modified GC-rich PCR. In each image, lane 23



M is for marker, and the last lane is the negative control. The reaction conditions of (a), (b), (c) are 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

as follows. The PCR protocol was: 5 minutes at 94 oC for pre-denaturation, followed by 35 cycles of: 45 s at 94 oC, 90 s at 64.2 oC, 60 s at 72 oC. Then the cycling was terminated after incubation at 72 o

C for 5 min. (a) PEI was added into the PCR mixture, and for lane 1 to lane 3, its final concentration

was 0, 14, 120 mg/L, respectively. (b) {(Au0)100-PEI-mPEG24} NPs was added into the PCR mixture, and for lane 1 to 4, its final concentration was 0, 0.32, 0.40, 0.44 mg/L, respectively. (c) {(Au0)200-PEI-mPEG24} NPs was added into the PCR mixture, and for lane 1 to 4, its final concentration was 0, 0.24, 0.32, 0.36 mg/L, respectively. (d) included two kinds of reaction conditions. Lane 1, 2 and 3 were generated under the above reaction condition. Lane 4 and 5 were generated under the follow reaction condition. The PCR protocol was: 5 minutes at 94 oC for pre-denaturation, followed by 35 cycles of: 25 s at 94 oC, 2 min at 64.2 oC, 3 min at 72 oC. Then the cycling was terminated after incubation at 72 oC for 5 min. {(Au0)300-PEI-mPEG24} NPs was added into the PCR mixture. For lane 1 to 5, its final concentration was 0, 0.20, 0.28, 0, 0.40 mg/L, respectively. Figure 8. The temperature variation curve of the solution containing the additives in PCR tubes.

24


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ACS Applied Materials & Interfaces NH2

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

NH 2

{(Au 0)100-PEI-mPEG24} NPs

NH 2 NH2 NH 2 NH2

NH 2 NH 2

NH2

NH 2

NH2 NH 2

NH 2

mPEG-COOH

HAuCl4

EDC/NHS

NaBH 4

{(Au 0)200-PEI-mPEG24} NPs NH 2

NH 2 NH2

NH 2

NH2 NH 2 NH 2

o NHC-mPEG

{(Au0) 300-PEI-mPEG24} NPs

NH2

Au NPs

NH2

Scheme 1

25



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

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Figure 1

26


Page 27 of 34

1.5 0

{(Au )100-PEI-mPEG24} NPs 0

{(Au )200-PEI-mPEG24} NPs

1.2

0


Absorbance

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


0.9 0.6 0.3 0.0 300

400

500

600

700

800

Wavelength (nm)

Figure 2

27



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Figure 3

28


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Figure 4

29



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Figure 5

30


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Figure 6

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Figure 7

32


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30 PEI 0 {(Au )100-PEI-mPEG24} NPs 0


28

0


o

Temperature ( C)

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


26 24 22 20

0

10

20

30

Time (s)

Figure 8

33



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TOC graphic

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Mechanistic Studies of Enhanced PCR Using PEGylated PEI

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