[
    {
        "id": 1,
        "chunk": "# Few-Layer Black Phosphorus as an Artificial Substrate for DNA Replication  \n\nJie Gui, Yunfei Bai, Huizhen Li, Jian Peng, Yufan Huang, Liping Sun,\\* and Jian Weng\\*  \n\nCite This: ACS Appl. Nano Mater. 2020, 3, 1775−1782",
        "category": " References"
    },
    {
        "id": 2,
        "chunk": "# ACCESS I Metrics & More  \n\nArticle Recommendations  \n\nSupporting Information  \n\nABSTRACT: DNA replication is the basis for biological inheritance in nature. The plasma membrane of prokaryotic bacteria might provide a supporting surface for the attachment of either polymerase or DNA during in vivo DNA replication. In order to understand that fixing DNA or polymerase on the membrane is important for highly efficient DNA replication, we develop a system to mimic prokaryotic DNA replication. We introduce a two-dimensional nanomaterial, few-layer black phosphorus (BP), which shares some similar properties with bacteria plasma membrane in dimension, surface group, surface charge, hydrophilicity, and thickness. Either polymerase or DNA primer is immobilized on a BP surface to initiate in vitro DNA replication. The results show that BP could promote DNA replication with high efficiency in both cases. The DNA yield is increased to more than 6 times and nonspecific products are reduced significantly. This discovery might deepen our understanding about the role of immobilization of the replication component on the membrane and application of BP as an artificial substrate for DNA replication. KEYWORDS: 2D nanomaterials, DNA replication, cell membrane, black phosphrous, biomimic  \n\n![](images/86f6c82fb8c7edd234ff9332f246b962f64d3e4a84290a9f073351e1961d95de.jpg)",
        "category": " Abstract"
    },
    {
        "id": 3,
        "chunk": "# INTRODUCTION  \n\nFew-layer black phosphorus (BP) is a two-dimensional (2D) nanomaterial widely used in the photoelectronic field because of its tunable band gap and anisotropic properties.1 In addition, BP has many biomedical applications, such as cancer imaging, cancer therapy, drug delivery, and various types of biosensing owing to its large surface area and excellent biocompatibility and biodegradability.2−5 The exposed lone pairs at the BP surface make phosphorus very reactive to oxygen, leading to the formation of negatively charged phosphate ions covered on its surface.6,7 Some strategies have been developed to enhance its stability. Xinget al. demonstrated that three-dimensional graphene oxide $(\\mathrm{GO})/$ BP hybrid aerogels exhibit excellent photothermal stability in ambient conditions.8 Yu’s group found that ${\\mathrm{Ag}}^{+}$ can be spontaneously adsorbed on the BP surface via cation $-\\pi$ interactions, rendering BP more stable in air.9 Although BP is unstable in water and air, its degradation products, phosphorus oxides, are nontoxic to the human body.  \n\nDNA replication is one of the most rapid and efficient processes that take place within cells. Genetic information is transferred from parent to progeny organisms by the faithful replication of parental DNA. The ability of cells to maintain a high degree of order depends on the accurate duplication of  \n\nDNA.10,11 Some researchers suggest that a bacteria plasma membrane might provide a supporting surface for DNA replication, and chromosomal DNA might be anchored on the bacterial membrane surface to form a DNA/membrane complex to initiate DNA replication. The origin of replication (oriC) of the Escherichia coli chromosome might bind with high affinity to the cell membrane.12,13 Similarly, the nuclear matrix (or skeleton) in eukaryotic cells could also provide a framework for efficient and precise genome duplication.14 The replication complexes might be attached to the nuclear matrix when DNA is replicated.15 Therefore, a supporting surface, such as plasma membrane or nuclear matrix, may be very important for the in vivo DNA replication process. Anionic phospholipids, such as phosphatidylglycerol and cardiolipin, would play a role in bacteria chromosomal replication by regulating the initiator protein DnaA. The activated initiator protein DnaA at the cell membrane recognizes and binds the oriC site of the bacterial chromosome to initiate DNA  \n\n![](images/868c00371c50f2ad9c7e9f4521a2ab12395f5e2bdb6af70e4408127142f8d1bf.jpg)  \nFigure 1. Comparison of the plasma membrane and BP. (a) The plasma membrane consists of a lipid bilayer. The phospholipid has a polar headgroup and two hydrophobic hydrocarbon tails. X represents a hydrogen or alcohol headgroup attached to the phosphate group of the  \n\nmembrane. (b) BP is composed of several layers of black phosphorene. Its surface is covered by phosphate ions. The yellow area represents the hydrophilic region, while the purple area represents the hydrophobic region. TEM images of the plasma membrane of $E_{\\sun}$ . coli $\\tt D H S\\alpha$ (c) and BP (d). (e) High-resolution P 2p XPS spectrum of BP. (f) $\\zeta$ potentials of the bacteria membrane $\\cdot{-}26.8~\\mathrm{mV})$ and BP $\\left(-30.4~\\mathrm{mV}\\right)$ . The contact angles of the bacteria plasma membrane of E. coli $\\mathtt{D H S}\\alpha$ $(\\mathbf{g})$ and BP (h). AFM curves and height profiles of the bacteria plasma membrane (i) and BP (j).  \n\nFigure 1. continued   \nTable 1. Similarities between the Plasma Membrane and BP   \n\n\n<html><body><table><tr><td></td><td> dimension</td><td> surface group</td><td>surface charge (§ potential), mV</td><td> hydrophilicity (contact angle), deg</td><td>thickness, nm</td></tr><tr><td>plasma membrane</td><td>2D</td><td> phosphate group</td><td>-26.8</td><td>16.1</td><td>4.0</td></tr><tr><td>BP</td><td>2D</td><td> phosphate group</td><td>-30.4</td><td>15.3</td><td>4.6</td></tr></table></body></html>  \n\nreplication.16,17 When anionic phospholipids are depleted, the chromosomal replication of E. coli is inhibited. Although a lot of effort has been expended to investigate the DNA/membrane or polymerase/membrane complex of the bacteria, it is still unclear how the membrane assists in DNA replication.18−22  \n\nCurrently, there are two putative models to explain the DNA replication mechanism: train and factory models. In the train model, the DNA template is fixed and DNA polymerase moves along the DNA template like a train on a track. In the factory model, the DNA polymerase is stationary and DNA is pulled through.23 In both models, the DNA polymerase or DNA might be attached to a fixed site to facilitate DNA replication, which remains elusive to date. In order to understand the role of fixing the polymerase or DNA on plasma membrane for DNA replication, we develop a simple system to mimic the replication site associated with the membrane. We introduce 2D BP as an artificial substrate to promote in vitro DNA replication.  \n\nAlthough the chemical structure and mechanical performance of BP are very different from that of the plasma membrane, BP also shares some similar properties, including (1) 2D planar structure, (2) surface phosphate-containing groups, (3) surface negative charge, (4) hydrophilic surface, and (5) nanoscale thickness (Figure 1). We immobilize either polymerase or DNA on the BP surface (Scheme S1) to replicate DNA. The results show that BP displays excellent capability to improve the efficiency of DNA synthesis by increasing the DNA yield to more than 6 times and reducing the nonspecific products significantly. It could be used as an artificial substrate for DNA replication and an efficient enhancer for in vitro DNA amplification techniques.",
        "category": " Introduction"
    },
    {
        "id": 4,
        "chunk": "# RESULTS AND DISCUSSION  \n\nSimilarities of the Plasma Membrane and BP. The planar phospholipid bilayer of the plasma membrane (Figure 1c) is a natural 2D material. BP also shows a similar 2D structure (Figure 1d,j). The surface of the plasma membrane is covered with phosphate-containing groups. The existence of a $_{\\mathrm{P-O}}$ bond resulting from BP oxidation is confirmed by X-ray photoelectron spectroscopy (XPS) spectrum (Figure 1e). The two strong peaks at 129.7 and $131.6\\ \\mathrm{eV}$ correspond to the $2\\mathrm{p}_{3/2}$ and $2\\mathsf{p}_{1/2}$ orbitals of zerovalent phosphorus, respectively. The weak peak at $133.9\\mathrm{eV}$ is the signal of oxidized phosphorus $\\left(\\mathrm{P-O}\\right)$ .24 The plasma membrane surface carries negative charges ( $-26.8\\mathrm{\\bar{\\mV}}$ because of the negatively charged phosphate group (Figure 1f). Similarly, the BP surface is also negatively charged $\\left(-30.4~\\mathrm{\\mV}\\right)$ , which originated from oxidized phosphorus compounds on its surface. The plasma membrane possesses a hydrophilic surface and a hydrophobic core, which is attributed to the hydrophobic fatty acid tails of the phospholipid buried in the interior and the phosphatecontaining hydrophilic headgroup exposed outside26 (Figure 1a). The contact angle $(\\theta)$ of the bacteria plasma membrane of E. coli is $16.1^{\\circ}$ (Figure ${\\mathrm{lg}}{\\mathrm{,}}$ ), confirming the hydrophilicity of the membrane surface. Likewise, the surface of BP is also hydrophilic $\\mathit{\\theta}=15.3^{\\circ}.$ ; Figure 1h). The thickness of the phospholipid bilayer measured as the phosphorus-to-phosphorus spacing is about $4.0\\ \\mathrm{nm},^{27}$ which is also confirmed by atomic force microscopy (AFM; Figure 1i). The AFM image indicates that the thickness of BP is $4.6\\pm1.2\\ \\mathrm{nm}$ (Figure 1j), which is close to the thickness of the plasma membrane. The above results suggest that BP and the plasma membrane are similar in their 2D structure, thickness, surface phosphate group, negative charge, and hydrophilic surface (Table 1).  \n\nImmobilization of DNA/Polymerase on the BP Surface. In order to investigate the role of fixation for DNA replication, first we immobilized DNA polymerase on the BP surface to initiate in vitro DNA replication in our system. Polymerase chain reaction (PCR) was chosen as a model reaction to evaluate the in vitro DNA amplification efficiency on the surface of BP because of its wide applications in biomedical fields, such as genomic studies, clinical diagnosis, and forensic identification. 28−31 Similar to the factory model, when DNA polymerase is fixed on the BP surface, DNA templates move through the polymerase (Scheme S1a). We immobilized $P f u$ polymerase on the BP surface by either physical adsorption or chemical bonding (Figure 2a,b). 2- Methylimidazole was used to activate the phosphate groups on BP.21 $P f u$ polymerase was conjugated with BP through the reaction between the amino group of polymerase and the phosphate group of BP. Fourier transform infrared spectra (FTIR) and XPS spectra confirmed the conjugation of ${P f u}$ polymerase with BP (Figure S1).  \n\nIn agarose gel electrophoresis, the target band intensity of the amplified products responds to the yield of DNA amplification. The smear or wrong bands located outside the target band position come from nonspecific amplification.22 We found that new DNA strands with a length of 689 bp were successfully synthesized. Both BP and $P f u$ complexes of physical adsorption or chemical bonding could enhance the DNA replication efficiency (Figure 2c). However, because of a sophisticated and time-consuming cross-linking process to obtain chemically bonded $\\mathrm{BP}/P f u$ polymerase, we used physically adsorbed $P f u$ polymerase in subsequent experiments.  \n\nAfter $P f u$ polymerase was physically adsorbed on BP, with increasing BP concentration $(0.02{-}0.32~\\mathrm{\\mg~\\mL^{-1}})$ , the intensity of the target band at 689 bp increased and the smear bands disappeared gradually (Figure 2d). At $0.08~\\mathrm{mg}$ $\\mathrm{mL}^{-1}$ BP, a maximum yield, which was over 6 times that of the control sample without BP (Figure S2), was reached. The yield and specificity of DNA replication increased with the BP concentration, suggesting that BP could promote DNA replication. Then we thoroughly investigated the effect of BP on DNA amplification at different conditions. We found that BP could improve both the specificity and yield of DNA replication in wide ranges of template DNA $(2{-}400~\\mathrm{pg}~\\mu\\mathrm{L}^{-1})$ , primer concentration $\\left(0.4\\mathrm{-}0.08\\mathrm{\\overset{\\sim}{m}M}\\right)$ , and annealing temperature $(25-65~^{\\circ}\\mathrm{C})$ (Figure $S3a,\\mathrm{b,e},$ ). Moreover, it reduced the amplification from 30 to 20 cycles and the extension time from 1 to $0.5\\mathrm{\\min}$ (Figure S3c,d). Thus, BP could significantly shorten the optimization time of in vitro DNA amplification, saving both time and cost. Besides promoting short-fragment amplification, BP can also help the amplification of longer target DNA (3015−5225 bp) and three-round DNA amplification (Figure S4). In addition, we also investigated the effect of BP on DNA amplification using different DNA polymerases. BP could significantly improve the specificity and yield of these polymerases (Figure S5). All of the above results show that BP could promote the DNA replication efficiency in multiple aspects.  \n\n![](images/01479bc715389d57606d1782dec3e030b966054e1809f5676a45020ee1ae0f16.jpg)  \nFigure 2. Fixation of DNA polymerase on BP to promote in vitro DNA amplification. (a) Schematic of $P f u$ polymerase adsorbed on the BP surface. (b) Schematic of $P f u$ polymerase fixed on the BP surface by chemical bonding. (c) DNA amplification using $P f u$ polymerase fixed on BP. (c) Control sample without BP: (1) physically adsorbed $P f u$ polymerase; (2) chemically bonded $P f u$ polymerase. The concentration of BP is $0.08\\mathrm{\\mg\\mL^{-1}}$ . (d) DNA amplification with $P f u$ polymerase adsorbed on BP. BP concentrations increase from 0.02 to $\\mathrm{0.32~mg~mL^{-1}}$ . M: DNA marker. C: control sample without BP. (e) Fluorescent spots of the amplified products in the supernatant and BP precipitation after centrifugation.  \n\nThe polymerase was fixed on the BP surface by electrostatic interaction because the isoelectric point of $P f u$ polymerase is 8.5; therefore, $P f u$ is positively charged in a buffer $\\left(\\mathrm{pH}~7.4\\right)$ and can be adsorbed onto a negatively charged BP surface. The $\\zeta$ potential of BP increased from $-24.9$ to $-9.39\\ \\mathrm{mV}$ after the addition of $P f u$ polymerase (Figure S6). This result shows that there is strong electrostatic interaction between BP and $P f u$ polymerase. The low dissociation constant $\\left(K_{\\mathrm{d}}=1.39\\times10^{-6}\\right.$ M) determined by isothermal titration calorimetry indicates a high affinity of $P f u$ polymerase with BP. The negative Gibbs free energy change $\\mathrm{'}\\Delta G=-32.35\\ \\mathrm{kJ\\moL^{-1}}$ ; Figure S7) also suggests spontaneous binding of Pfu polymerase with BP.32 AFM showed that $P f u$ polymerase molecules were adsorbed onto BP (Figure S8). The height of $\\mathrm{BP}/P f u$ increased from 4.6 nm of BP to $10.5\\ \\mathrm{~nm}$ of $\\mathbb{B P}/P f u$ . Bicinchoninic acid measurement confirmed that $69\\%$ of Pfu polymerase was adsorbed onto the BP surface (Figure S9). A fluorescent spots study was performed to investigate whether DNA replication might occur on the BP surface. We used SYBR green I to dye the amplification product at different replication cycles because the fluorescent dye SYBR green I can enter the minor groove of double-stranded DNA (dsDNA).33 The spots of BP precipitation after centrifugation at 1−15 cycles were much brighter than those of the supernatant without BP, indicating that DNA amplification of the initial 15 cycles would occur on the surface of BP (Figure 2e and Table S6).  \n\nReal-time PCR (RT-PCR) was performed to monitor the amplification of a targeted DNA molecule during the amplification process. The threshold cycle $\\left(C_{\\mathrm{T}}\\right)$ represents the PCR cycle at which the fluorescent signal passes the fixed threshold. At the same initial concentration of template DNA, a smaller $C_{\\mathrm{T}}$ value indicates that the amplified DNA products can reach the threshold more quickly. The $C_{\\mathrm{T}}$ values decreased gradually after the ddition of BP, suggesting that BP would facilitate DNA amplification (Figure S10a). The melting temperature $\\left(T_{\\mathrm{m}}\\right)$ is the temperature at which the dsDNA strand separates. As the BP concentration increases, $T_{\\mathrm{m}}$ decreases from 73.8 to $71.4~^{\\circ}\\mathrm{C},$ indicating that BP might enhance the separation of dsDNA. Melting curve analysis indicated that BP decreased the $T_{\\mathrm{m}}$ value of dsDNA and accelerated the dsDNA denaturation process to obtain more single-stranded DNA (ssDNA; Figure S10b), which would benefit DNA replication on the surface of BP. The oxidized phosphorus compound could bind the $-\\mathrm{NH}$ or $\\scriptstyle{\\mathrm{C}}={\\mathrm{O}}$ groups of the exposed bases of ssDNA by hydrogen bonding between $\\scriptstyle\\mathrm{P=O\\cdotN\\bar{H}}$ or $\\mathrm{P-OH\\cdotO=C}$ , resulting in strong interaction between ssDNA and BP.34  \n\nImmobilization of a DNA Primer on the BP Surface. On the other hand, $\\mathsf{A l}^{3+}$ -modified BP $\\left(\\mathrm{BP-Al}^{3+}\\right)$ was used to fix the ${\\boldsymbol{5}}^{\\prime}$ -thiolated forward primer on the BP surface by $_{\\mathrm{Al}-S}$ bonding. Fluorescent intensity measurements confirmed that the DNA primer was immobilized on the ${\\mathrm{BP}}{\\cdot}{\\mathrm{Al}}^{3+}$ surface (Figure S11). This system could represent the train model because the DNA primer is fixed on the BP surface and the polymerase moves along the DNA template (Figure 3a).  \n\nThe new DNA products were amplified on the BP surface, and the yield increased gradually with increasing concentration of the BP- $\\mathbb{A}^{3+}$ -F1 DNA primer (Figure 3b). The DNA yield with BP was obviously higher than that of the control sample without BP (Figure 3c). A fluorescent spots study of 1−30 cycles showed that the precipitation of BP exhibited much stronger fluorescence than the supernatant, indicating that DNA replication would occur on the BP surface (Figure 3d). These results show that fixation of either the polymerase or the DNA primer could be effective in promoting in vitro DNA replication on the BP surface.  \n\nEffect of the Five Properties of BP on in Vitro DNA Amplification. In order to understand the importance of the five factors of membrane for DNA amplification, we changed the 2D dimension, surface phosphate group, surface charge, hydrophilicity, and thickness of BP, respectively, and compared their effects on in vitro DNA amplification. First, we used $\\mathrm{H}_{2}\\mathrm{O}_{2}$ as the oxidant to destroy the 2D structure of BP.  \n\n![](images/acda4cac22769111d3ee0b2efcea0209fa9131f7f7f5f37d6495a76b72d3e0fb.jpg)  \nFigure 3. BP promotes in vitro DNA amplification by immobilization of the DNA primer on the $\\mathrm{BP\\mathrm{\\cdot}A l^{3+}}$ surface. (a) Schematic of DNA replication on BP with a fixed forward primer. The forward DNA primer (black) is fixed on the BP surface. (b) Effect of the concentration of F1 DNA primer fixed on the BP surface upon DNA amplification. ${\\mathrm{BP}}{\\cdot}{\\mathrm{Al}}^{3+}$ was incubated with thiolated F1 DNA primer and centrifuged to remove unbound primers. The precipitate (BP$\\mathbb{A}^{3+}$ -F1 primer) was collected and redispersed in water. A total of $_{0-5}$ $\\mu\\mathrm{L}$ of BP- $\\mathbf{\\cdotAl}^{3+}$ -F1 primer was added to the amplification system. (c) DNA amplification using DNA fixed on BP. M: DNA marker. C: control sample with BP. The primers were not fixed on BP. 1: F1 DNA primer fixed on BP. (d) DNA amplification occurs on the BP surface supported by a fluorescent spots study. The concentration of BP and ${\\mathrm{B}}{\\bar{\\mathrm{P}}}{\\cdot}{\\mathrm{Al}}^{3+}$ is $\\mathrm{0.08~mg~mL^{-1}}$ .  \n\nTransmission electron microscopy (TEM) images showed that the 2D structure of BP was damaged by $\\mathrm{H}_{2}\\mathrm{O}_{2}$ oxidation (Figure 4a). XPS spectra suggested that the oxidation degree of BP increased with increasing concentration of $\\mathrm{H}_{2}\\mathrm{O}_{2}$ (Figure S12). The DNA replication efficiency decreased with increasing oxidation degree of BP (Figure 4a), confirming the important role of the 2D structure of BP in promoting DNA replication.  \n\nSecond, we used sodium dodecyl sulfate (SDS) with a sulfonic group and poly(acrylic acid) (PAA) with a carboxyl group to modify the surface of BP (Figures 4b and S13). We found that BP with phosphate groups promotes in vitro DNA amplification, while BP/SDS and BP/PAA inhibited DNA replication (Figure 4b). We also modified BP with sodium monododecyl phosphate (SDP) and found that it inhibited DNA replication (Figure S14, band 2), indicating that both the phosphate group and BP sheets play important roles in promoting PCR. Althought SDS and SDP contain phosphate groups, their hydrophobic dodecyl chains can penetrate the hydrophobic protein core, thus disrupting the tertiary and secondary structures of $P f u$ polymerase.35 As a result, DNA replication was inhibited. The exact function of phosphate groups for DNA replication remains unclear. Modifying different nanomaterials with phosphate functional groups might help to explain the mechanism for BP-promoted DNA replication.  \n\nThird, we investigated the effect of charge on DNA amplification, while negative surface charge is another common property of BP and the plasma membrane. We used neutral poly(ethylene glycol) (PEG), positively charged lysine, and chitosan to modify BP. For neutral PEG-modified BP (Figure  \n\n4c, band 2), the DNA replication efficiency decreased compared with that of negatively charged BP (band 1). After lysine and chitosan modification, the $\\zeta$ potentials of modified BP increased from $-30.4~\\mathrm{mV}$ of BP to $-6.9\\ \\mathrm{mV}$ of BP/lysine and $16.8\\ \\mathrm{mV}$ of BP/chitosan, respectively (Figure $\\mathsf{4c})$ , and DNA replication was completely inhibited (Figure 4c, bands 3 and 4). Thus, negative surface charge plays an important role in DNA replication. The effect of negative charge on DNA replication still needs further investigation, such as applying an electric field to control the surface charge of BP.  \n\nTo understand that the hydrophilic surface is also important for DNA amplification, BP was modified with weakly hydrophobic $\\mathrm{\\hat{(}C H_{3}C H_{2})_{2}N C H_{2}-}$ (DEMA) and strongly hydrophobic 2-naphthalenethiol (2-NAT). The contact angles of modified BP increased from $15.3^{\\circ}$ of BP to $50.1^{\\circ}$ of BP/ DEMA and $119.2^{\\circ}$ of BP ${\\bf-A l}^{3+}2\\mathrm{NAT}$ (Figure $\\mathrm{4d}\\`_{,}$ ). The result shows that DNA amplification was also completely inhibited by hydrophobic BP/DEMA and ${\\mathrm{BP}}{\\cdot}{\\mathrm{Al}}^{3+}2{\\mathrm{NAT}}$ (Figure 4d, bands 2 and 3). These results suggest that the hydrophilic BP surface also makes an important contribution to DNA replication.  \n\nFinally, we explored the effect of the BP thickness on DNA replication. We prepared BP with different thicknesses (86.3, 42.4, and $4.8~\\mathrm{nm}$ , respectively; Figures $_{4\\mathrm{e}}$ and S15). BP with a thickness $(4.8~\\mathrm{nm})$ close to that of the plasma membrane (4.0 nm) has the best chance to promote DNA replication (Figure 4e, band 3). The above studies demonstrated that destroying any of the five aspects of BP would reduce the DNA replication efficiency. Therefore, all five properties are important for DNA replication. Despite the similarities of BP with the plasma membrane, the following aspects still need further investigation. The phospholipid membrane is permeable, and therefore part of the DNA polymerase or DNA could insert within the membrane, which is not possible in BP. Second, the phospholipid membrane is soft and has a potential curvature to its interface, while BP is a flat and rigid sheet. Finally, the lipid molecules are mobile and can diffuse in the plasma membrane, therefore changing the properties of the local DNA polymerase when they adsorb to its surface. Further study is required to investigate whether these properties are important in the replication process and how they contribute to the amplification rate.  \n\nHere we also compared BP with several other nanomateri$\\mathrm{als}^{36-38}$ owning part of the five similarities (Figure $S16\\mathrm{a}-\\mathrm{c}$ and Table S7). GO has no surface phosphate group. Few-layer boron nitride (BN) sheets are hydrophobic and possess neither a surface phosphate group nor a negative charge. Gold nanoparticles (GNPs) are zero-dimensional (0D) nanomaterials with no surface phosphate group. At a fixed concentration $(0.08~\\mathrm{mg~mL^{-1}}),$ , BP showed the best performance, followed by GNPs, GO, and BN (Figure S17a). We explored the optimized concentrations of GO, BN, and GNPs for DNA amplification (Figure $\\mathrm{S16d-f)}$ . Although all of them showed enhanced efficiency, BP displayed the strongest amplification without any smears, indicating its superiority over other nanomaterials (Figure S17b). BP had the lowest $C_{\\mathrm{T}}$ values in both fixed and optimal concentrations (Figure S17c,d), which suggests that BP would accelerate DNA amplification. BP is more biocompatible than the other nanomaterials because of its similar structure to the plasma membrane (Figure 1a). Another unique advantage of BP is that it can be degraded into biocompatible phosphorus oxides without any residual. However, GO, BN, and GNPs are all nondegradable materials;  \n\n![](images/cc29f2848ae12571dffefc114e790cdee25f34faa30af61e9b5a17c6c967c607.jpg)  \nFigure 4. Effect of the five properties of BP on in vitro DNA amplification. (a) BP with intact 2D structure promotes DNA amplification. The 2D dimension of BP is damaged by $\\mathrm{H}_{2}\\mathrm{O}_{2}$ . Samples $_{1-7}$ are amplification products with BP oxidized by 0, 0.01, 0.02, 0.04, 0.05, 0.06, and $0.07\\%$ $\\mathrm{H}_{2}\\mathrm{O}_{2},$ respectively. The TEM images show the damaged structure of BP. (b) BP with phosphate groups promotes in vitro DNA amplification: (1) BP $\\mathrm{(P\\bar{O}_{4}{}^{3-})}$ ; (2) BP/SDS $(\\mathrm{SO}_{3}\\mathrm{^{-}})$ ; (3) BP/PAA $(\\mathrm{COO^{-}})$ . (c) Negatively charged BP promotes in vitro DNA amplification: (1) BP $(-)$ ; (2) BP/PEG (neutral); (3) BP/lysine $\\left(+\\right)$ ; (4) BP/chitosan $\\left(+\\right)$ . $\\zeta$ potentials of BP, BP/PEG, BP/lysine, and BP/chitosan are $-30.4_{;}$ $-20.2$ , $-6.9,$ and $16.8~\\mathrm{mV},$ respectively. (d) BP with hydrophilic surface promotes in vitro DNA amplification: (1) BP (hydrophilic); (2) BP/DEMA (weakly hydrophobic); (3) ${\\mathrm{BP}}{\\cdot}{\\mathrm{Al}}^{3+}2{\\mathrm{NAT}}$ (strongly hydrophobic). The contact angles of BP, BP/DEMA, and B ${\\mathrm{.P}}{\\mathrm{-}}{\\mathrm{Al}}^{3+}2{\\mathrm{N}}{\\mathrm{AT}}$ are $15.3^{\\circ}$ , $50.1^{\\circ};$ , and $119.2^{\\circ}.$ respectively. (e) BP with smaller thickness promotes DNA replication. The thicknesses of BP in samples 1−3 are 86.3, 42.4, and $4.8\\ \\mathrm{nm},$ respectively. M: DNA marker. C: control sample without BP. The concentrations of BP and modified BP are both $0.08~\\mathrm{mg~mL^{-1}}$ .  \n\nthus, an additional purification process is required to remove these materials, which could be harmful for subsequent transformation, transcription, or sequencing procedures. Compared with 0D BP quantum dots and soft BP hydrogels composed of agarose and PEGylated BP sheets, which have been used in cancer therapy and imaging,2,39 few-layer BP sheets mimic the 2D plasma membrane and are more suitable as artificial substrates for DNA replication. Although BP has negligible cytotoxicity, its instability has limited its commercialization. Strategies to enhance the ambient stability of BP and prolong its preservation time need to be developed to promote the biomedical applications of BP. For example, Lei et al. offers an efficient approach to preventing BP sheets from oxidation by adatom decoration on BP, which could significantly shift their conduction-band minimum below the $\\mathrm{O}_{2}/\\mathrm{O}_{2}^{-}$ redox potential.40  \n\nThe above results suggest that mimicking all five properties of the plasma membrane might be important to promote in vitro DNA replication. Meanwhile, we also sequenced the amplification products in the absence or presence of BP. The sequence alignment showed no difference. One example of the 4024 bp amplified product sequencing is given in Figure S18. The result indicates that BP could not only improve DNA replication but also maintain the fidelity of $P f u$ polymerase.  \n\nDNA Amplification of Clinical Samples with BP. For sophisticated DNA samples extracted from clinical samples or multiple-round $\\operatorname{PCR},$ nonspecific DNA fragments and low yield remain a problem. We investigated the effect of BP on human mitochondrial DNA amplification. Figure S19 shows that BP improved the efficiency of three-round PCR and could amplify long DNA fragments. These results indicate that BP could also be used to improve the replication efficiency of clinical DNA samples.",
        "category": " Results and discussion"
    },
    {
        "id": 5,
        "chunk": "# CONCLUSIONS  \n\nOur study demonstrated that BP shares five similar properties with a phospholipid plasma membrane, including its 2D dimension, surface group, surface charge, hydrophilicity, and thickness. Fixing DNA or polymerase on the BP surface could promote DNA replication with high efficiency. This study opened up more opportunities for the application of BP as an artificial substrate for DNA replication and an efficient enhancer for in vitro DNA amplification techniques.",
        "category": " Conclusions"
    },
    {
        "id": 6,
        "chunk": "# ASSOCIATED CONTENT",
        "category": " References"
    },
    {
        "id": 7,
        "chunk": "# $\\bullet$ Supporting Information  \n\nThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsanm.9b02456.  \n\nMaterials and methods, schematic view of DNA replication models on the surface of BP, characterization of the $\\ensuremath{\\mathrm{BP}}\\cdot\\ensuremath{\\boldsymbol{P}}\\ensuremath{\\boldsymbol{f u}}$ complex, effect of BP on PCR, measurements of BP and $P f u$ polymerase interaction, calculation of the maximum amount of DNA molecules on BP and the amount of DNA obtained at each PCR cycle, RT-PCR, fluorescent intensity measurement of ssDNA-modified BP, XPS spectra of BP with different oxidation degrees, FTIR spectra of SDS- and PAAmodified BP, effect of SDP-modified BP on $\\mathrm{PCR},$ AFM images of BP with different thicknesses, comparison of the effects of different nanomaterials on $\\mathrm{PCR},$ effect of BP on the fidelity of $P f u$ polymerase, and effect of BP on PCR of DNA isolated from clinical samples (PDF)",
        "category": " Materials and methods"
    },
    {
        "id": 8,
        "chunk": "# AUTHOR INFORMATION",
        "category": " References"
    },
    {
        "id": 9,
        "chunk": "# Corresponding Authors  \n\nLiping Sun − Key Laboratory of Biomedical Engineering of Fujian Province, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, P. R. China; $\\circledcirc$ orcid.org/0000-0001-8707-7446; Phone: $+86-592$ - 2183181; Email: sunliping@xmu.edu.cn Jian Weng − Key Laboratory of Biomedical Engineering of Fujian Province, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, P. R. China; $\\circledcirc$ orcid.org/0000-0002-5813-6061; Phone: $+86-592$ - 2183181; Email: jweng@xmu.edu.cn  \n\nAuthors Jie Gui − Key Laboratory of Biomedical Engineering of Fujian Province, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, P. R. China; $\\circledcirc$ orcid.org/ 0000-0001-8562-6990 Yunfei Bai − Key Laboratory of Biomedical Engineering of Fujian Province, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, P. R. China Huizhen Li − Key Laboratory of Biomedical Engineering of Fujian Province, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, P. R. China; $\\circledcirc$ orcid.org/0000-0002-1773-6749 Jian Peng − Key Laboratory of Biomedical Engineering of Fujian Province, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, P. R. China Yufan Huang − Department of Breast Surgery, The First Affiliated Hospital of Xiamen University, Xiamen 361003, P. R. China  \n\nComplete contact information is available at: https://pubs.acs.org/10.1021/acsanm.9b02456",
        "category": " References"
    },
    {
        "id": 10,
        "chunk": "# Author Contributions  \n\nL.S. and J.W. designed the experiments, performed data interpretation, and wrote the manuscript. J.G., Y.B., H.L., and J.P. performed the experiments and analyzed the data. Y.H. collected clinical samples.",
        "category": " References"
    },
    {
        "id": 11,
        "chunk": "# Funding  \n\nThis work was supported by the National Natural Science Foundation of China (Grants 81872415 and 81571764).",
        "category": " References"
    },
    {
        "id": 12,
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