碳納米管在作物研究中的應(yīng)用進(jìn)展與分析

劉學(xué)文，高軍濤，李 赫，楊自尚，陳 靜

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碳納米管在作物研究中的應(yīng)用進(jìn)展與分析

劉學(xué)文1，高軍濤2，李 赫1，楊自尚1，陳 靜1※

（1. 河南農(nóng)業(yè)大學(xué)機(jī)電工程學(xué)院，鄭州 450002；2. 中電科新防務(wù)技術(shù)有限公司，鄭州 450047）

碳納米管在農(nóng)作物研究中的應(yīng)用對(duì)象已經(jīng)涵蓋了大米、玉米、小麥、番茄、葡萄等常見的農(nóng)作物。由于研究目的不同，使用的碳納米管種類、分散方式、濃度也不同，導(dǎo)致文獻(xiàn)中出現(xiàn)了試驗(yàn)結(jié)果多樣化的問題。為了建立一個(gè)較為統(tǒng)一的碳納米管試驗(yàn)框架，該研究首先以作物培育、抗逆、生理指標(biāo)監(jiān)測和農(nóng)業(yè)基因工程為綱，對(duì)碳納米管在作物研究中的應(yīng)用進(jìn)展進(jìn)行了綜述，并歸納了文獻(xiàn)中使用的碳納米管參數(shù)及其應(yīng)用效果。通過綜合分析，論證了作物研究中存在碳納米管有效濃度未知、特異性作用不明顯、間接生物毒性研究不足的問題，并針對(duì)性地給出了作物研究中的碳納米管試驗(yàn)方案建議，包括碳納米管的選型、培養(yǎng)基的制備以及表征方法。最后，與以往利用原始和共價(jià)修飾的碳納米管進(jìn)行作物培育不同，在未來，利用非共價(jià)修飾的碳納米管或碳納米管的非共價(jià)堆積作用進(jìn)行作物研究是重要的發(fā)展方向。

納米；作物；碳納米管；表面修飾；作物培育；生物毒性

0 引 言

碳納米管（carbon nanotube，CNT）因管壁層數(shù)不同而分為單壁碳納米管（single-walled carbon nanotube，SWCNT）和多壁碳納米管（multi-walled carbon nanotube，MWCNT），其優(yōu)良的光學(xué)特性、生物相容性和尺寸特性已經(jīng)被廣泛認(rèn)可并被應(yīng)用于許多領(lǐng)域[1-2]。在結(jié)構(gòu)上，SWCNT可以被視為由石墨烯沿某一矢量方向卷曲而成的單層管狀結(jié)構(gòu)，直徑0.4～2 nm（更大直徑的SWCNTs在技術(shù)上是可行的，但容易導(dǎo)致缺陷甚至表面塌陷），長度一般在微米到亞毫米量級(jí)；MWCNT可以被視為多個(gè)不同直徑的SWCNTs的同軸嵌套形式，層數(shù)為幾層到幾十層不等。各管層通過范德瓦爾斯力分開，管層間距為0.34～0.41 nm[3]。MWCNT的外直徑2～100 nm，內(nèi)直徑1～3 nm，長度一般在微米到亞毫米量級(jí)。也有報(bào)道稱可以制備長度為厘米尺度的SWCNTs[4-5]和MWCNTs[6-8]。

近年來，CNTs與農(nóng)作物相互作用已經(jīng)成為研究熱點(diǎn)，主要集中在CNTs對(duì)作物生長發(fā)育的促進(jìn)和生物毒性等方面[9-10]，目標(biāo)物種已經(jīng)涵蓋了大米、玉米、小麥、番茄、葡萄等常見的農(nóng)作物。植物的生長發(fā)育是個(gè)非常復(fù)雜的過程，受眾多內(nèi)在外在因素的影響，因此文獻(xiàn)中關(guān)于CNTs影響作物生長發(fā)育的形式具有多樣性的特點(diǎn)，其中積極作用包括提高種子發(fā)芽率，增大芽長和根長，增加葉片的數(shù)量，增加花和果實(shí)數(shù)量以及提高應(yīng)對(duì)脅迫環(huán)境的能力等；但當(dāng)CNTs濃度高時(shí)，也會(huì)對(duì)作物生長產(chǎn)生氧化損傷、發(fā)芽率降低等負(fù)面影響[11]。因此，有必要對(duì)文獻(xiàn)中的農(nóng)作物的種類、生長階段、培育方式以及碳納米管的種類、分散方式、施用方法、濃度進(jìn)行總結(jié)歸納，以期發(fā)現(xiàn)規(guī)律、為后續(xù)研究者建立一個(gè)較為統(tǒng)一的研究框架。最后，本文也探討了CNTs在作物研究中可能的應(yīng)用范圍以及發(fā)展趨勢。

1 作物對(duì)碳納米管的吸收

植物細(xì)胞壁是限制外源性生物分子遞送到植物體內(nèi)的屏障，由于細(xì)胞壁孔隙因植物種類、組織類型不同而不同，所以外源性分子穿過細(xì)胞壁的難易程度和通量也不同。按照現(xiàn)有的數(shù)學(xué)模型[12-13]，直徑超過20 nm的納米材料進(jìn)入植物是非常困難的[14]。

但許多文獻(xiàn)報(bào)道了大尺寸CNTs可以穿過植物細(xì)胞壁，SMIRNOVA等發(fā)現(xiàn)超聲分散的外徑（outside diameter，OD）20～70 nm，內(nèi)徑（inner diameter，ID）5～10 nm，長度≥2 μm的MWCNTs分散液可以進(jìn)入Onobrychis arenaria的根部和葉部[15]。WILD等使用超聲分散的外徑110～170 nm（長度≤9 μm）的MWCNTs分散液水培小麥時(shí)發(fā)現(xiàn)MWCNTs可以刺穿表皮和根毛細(xì)胞壁，貫穿整個(gè)根部的伸長區(qū)、根毛和分枝以及根冠，MWCNTs進(jìn)入細(xì)胞質(zhì)的深度約為4 μm。延長培育時(shí)間（最長28天）會(huì)導(dǎo)致穿透細(xì)胞壁的MWCNTs數(shù)量增加，但進(jìn)入的深度并未超過4 μm[16]。KHODAKOVSKAYA等發(fā)現(xiàn)平均直徑20 nm，長度0.5～1 μm的MWCNTs可以進(jìn)入煙葉細(xì)胞[17]。

因此，CNTs在細(xì)胞壁處的積累可能改變了細(xì)胞壁的性質(zhì)，主要有以下幾種途徑：

1）CNTs在細(xì)胞壁和角質(zhì)層上造成了新的、更大的孔隙，并在纖維微絲中產(chǎn)生破裂和破壞，最終促進(jìn)了更大尺寸CNTs的吸收[18]。

2）CNTs會(huì)誘導(dǎo)活性氧（reactive oxygen species，ROS）大量爆發(fā)[19]，其中，OH自由基能夠在植物多糖中裂解糖鍵，引起細(xì)胞壁木糖葡聚糖聚合物的分裂，導(dǎo)致植物細(xì)胞壁松動(dòng)，氣孔增大[20]。類似地，環(huán)境脅迫誘導(dǎo)的ROS爆發(fā)也會(huì)增加對(duì)大尺寸納米顆粒（nanoparticles，NPs）的吸收[21-22]。

3）表面修飾也可以輔助大尺寸CNTs穿透細(xì)胞壁。例如，Serag等驗(yàn)證了用纖維素酶修飾過尖端和側(cè)壁的杯狀堆疊碳納米管（直徑范圍60～100 nm）可以穿過擬南芥細(xì)胞壁，并認(rèn)為是通過修飾于CNTs表面的纖維素酶和細(xì)胞壁之間的局部接觸位點(diǎn)進(jìn)入的，其中，纖維素的局部水解起到了輔助作用[23]。

雖然作物對(duì)CNTs的吸收已被試驗(yàn)驗(yàn)證，但CNTs穿透作物細(xì)胞壁的原理及其在細(xì)胞間轉(zhuǎn)運(yùn)的動(dòng)力學(xué)原理尚缺乏透徹研究。

2 碳納米管在作物研究中的應(yīng)用

不同修飾物修飾的CNTs在作物研究中的應(yīng)用（如圖 1所示）也不同：1）未修飾的或表面羧基化的CNTs主要用來研究對(duì)作物培育的影響，羧化的CNTs比未修飾的CNTs具有更優(yōu)的可移動(dòng)性，使得芥菜種子發(fā)芽更快、發(fā)芽率更高[24]；2）生物大分子（如蛋白質(zhì)、核酸[25]等）修飾的或聚合物（如殼聚糖、聚乙二醇PEG、聚乙烯亞胺PEI、聚氨基胺PAMAM[26]等）與生物大分子共同修飾的CNTs常被用來研制生物傳感器[27]或進(jìn)行DNA/RNA遞送等；3）表面活性劑（如月桂醇硫酸鈉SDS、膽酸鈉SC[28]、脫氧膽酸鈉SDC等）或單純聚合物[29]修飾的CNTs對(duì)農(nóng)作物影響鮮有報(bào)道。下面是碳納米管在作物研究中具體應(yīng)用實(shí)例的詳細(xì)介紹。

圖1 碳納米管在作物研究中的應(yīng)用

2.1 碳納米管在作物培育中的應(yīng)用

CNTs的作用包括[30-32]：提高種子發(fā)芽率，增加芽長和根長，增加根和葉片的數(shù)量，增加花和果實(shí)數(shù)量，增強(qiáng)光合作用率，提高蒸騰速率，增加作物的生物質(zhì)，增強(qiáng)水和礦物質(zhì)的攝取以及提高轉(zhuǎn)運(yùn)速率等。

2.1.1 碳納米管對(duì)作物種子萌發(fā)的影響

2.3～23 mg/L CNTs處理過的芥菜種子比蒸餾水處理過的發(fā)芽率更高，50%的種子萌芽所需時(shí)間縮短了44%，而46 mg/L的CNTs明顯出現(xiàn)生物毒性[24]；SMIRNOVA等使用0.1～10 mg/L未修飾的MWCNTs分散液浸泡萌發(fā)Onobrychis arenaria種子，種子的發(fā)芽率有明顯提升，根和莖的長度均明顯增加[15]。Haghighi等用10～40 mg/L未修飾的MWCNTs（直徑8～15 nm，長度>10 μm）分散液直接萌發(fā)種子，發(fā)現(xiàn)番茄、洋蔥和小蘿卜的平均發(fā)芽時(shí)間延長；根長和莖長增加；40 mg/L的CNTs對(duì)洋蔥和小蘿卜產(chǎn)生生物毒性[33]。Ca?as等發(fā)現(xiàn)未修飾的SWCNTs會(huì)抑制番茄的根伸長，增強(qiáng)洋蔥和黃瓜的根伸長，PABS-CNTs會(huì)抑制生菜的根系伸長，而卷心菜和胡蘿卜不受上述兩種SWCNTs的影響[29]。

CNTs對(duì)種子萌發(fā)的影響呈現(xiàn)明顯的濃度依賴和種類依賴，低濃度的CNTs對(duì)種子的萌發(fā)產(chǎn)生積極影響。

2.1.2 碳納米管對(duì)作物幼苗生長的影響

Tiwari等利用磁力攪拌的MWCNTs-瓊脂混合物（CNTs直徑6～9 nm，長度5 μm）培養(yǎng)玉米種子，發(fā)現(xiàn)MWCNTs使黑色層種皮和根部細(xì)胞穿孔，因而水的輸送增強(qiáng)。在正常培養(yǎng)基中，低劑量MWCNTs被認(rèn)為是有益的，可改善根部吸水性、植物生物量和必需的Ca、Fe營養(yǎng)素的濃度，但在高濃度下抑制了玉米幼苗的生長[34]。賽鬧汪青等使用1 000 mg/L的MWCNTs培育水稻幼苗發(fā)現(xiàn)直徑30 nm以下的MWCNTs抑制了水稻幼苗的生長，但直徑大的影響更小，而直徑大于50 nm的MWCNTs卻顯著優(yōu)化了水稻幼苗的生長[35]。

CNTs對(duì)幼苗生長的影響存在濃度依賴，同時(shí)也存在直徑尺寸依賴，參照CNTs影響種子萌發(fā)的試驗(yàn)也可以發(fā)現(xiàn)：使用大直徑CNTs的文獻(xiàn)中，較少提及CNTs對(duì)作物培育的毒性。

2.1.3 碳納米管對(duì)花和果實(shí)數(shù)量的影響

在補(bǔ)充MWCNTs的土壤中種植長春花，MWCNTs通過誘導(dǎo)早期花卉發(fā)育和更高的花卉產(chǎn)量來刺激植物繁殖系統(tǒng)。在補(bǔ)充MWCNTs（濃度50? mg/L，200? mg/L，OD 13～18? nm，長度1～12 ?μm）和石墨烯的土壤中種植的長春花，總花產(chǎn)量分別提高了37%和58%[36]。在補(bǔ)充表面弱羧化MWCNTs的土壤中生長的番茄植物產(chǎn)生的花朵和果實(shí)是對(duì)照土壤的兩倍[37]。González-G等發(fā)現(xiàn)MWCNTs（OD 30～50 nm，長度10～20 μm）增加了番茄作物的果實(shí)產(chǎn)量和干枝生物量。由于抗壞血酸、類黃酮的含量和谷胱甘肽過氧化物酶的活性增加，抗氧化系統(tǒng)得到改善。碳納米管的應(yīng)用也提高了凈光合作用和水分利用效率[38]。包興成等發(fā)現(xiàn)4～12 mg/L的羧化MWCNTs（OD 20～30 nm，長度10～30μm）對(duì)夏黑葡萄的生長和果實(shí)品質(zhì)均有促進(jìn)作用，其中，8? mg/L的CNTs作用效果最佳[39]。

與前述對(duì)種子萌發(fā)的影響不同，上述文獻(xiàn)中并未發(fā)現(xiàn)高濃度的CNTs對(duì)種子萌芽率、根長、芽長、花和果實(shí)數(shù)量出現(xiàn)抑制作用。

2.1.4 碳納米管對(duì)作物培育的負(fù)面作用機(jī)理

CNTs會(huì)引起植株內(nèi)ROS過量爆發(fā)，導(dǎo)致植株氧化損傷。例如：Tan等發(fā)現(xiàn)用CNTs培養(yǎng)的水稻細(xì)胞ROS增加，細(xì)胞活力降低[40]。MWCNTs與草甘膦聯(lián)合的水培擬南芥的根系和地上部的生長受到顯著影響，丙二醛含量高，代謝抗氧化分子上調(diào)表明MWCNTs與草甘膦聯(lián)合施用引起強(qiáng)烈的氧化損傷，抗氧化酶活性的降低表明ROS與抗氧化防御系統(tǒng)之間由于ROS的不斷產(chǎn)生而失衡[41]。

CNTs促進(jìn)細(xì)胞壁非流動(dòng)性Ca2+流失，將引起后續(xù)植物損傷：Tiwari等研究發(fā)現(xiàn)CNTs與Fe2+同時(shí)存在時(shí)，細(xì)胞內(nèi)Fe2+的含量增加，而Ca2+的含量降低[34]。另外，CNTs可以將Fe3+還原成Fe2+，F(xiàn)e2+會(huì)繼續(xù)與Ca2+發(fā)生交換[42]。由于根部Ca2+主要固化于細(xì)胞壁，因此，CNTs的存在會(huì)加重細(xì)胞壁Ca2+的流失，可能造成根部細(xì)胞壁被穿孔，體內(nèi)營養(yǎng)物質(zhì)外滲，更易被病菌侵染等后果。

文獻(xiàn)中CNTs的作用結(jié)果因作物種類、CNTs類型和濃度的不同而不同，甚至出現(xiàn)相互矛盾的現(xiàn)象。通過分析CNTs產(chǎn)生負(fù)面作用的原理發(fā)現(xiàn)，當(dāng)試驗(yàn)中的培養(yǎng)基、培養(yǎng)方式、作物生長特性不同時(shí)，產(chǎn)生差異性結(jié)果是必然的。表1給出了典型文獻(xiàn)中的CNTs參數(shù)?？傮w來看，大直徑的、短型的、合適濃度的CNTs對(duì)作物的培育能夠起到積極作用。

2.2 碳納米管在作物對(duì)抗脅迫環(huán)境中的應(yīng)用

Martínez等發(fā)現(xiàn)MWCNTs在鹽脅迫下可以進(jìn)入成株的細(xì)胞，積累量較高，認(rèn)為MWCNTs對(duì)NaCl處理植物生長的積極影響是水分吸收增加的結(jié)果[30]。Pandey等發(fā)現(xiàn)MWCNTs減少了NaCl引起的毒性作用，鹽脅迫條件下栽培的作物長期施用MWCNTs改善了期望的顯性性狀：長春花發(fā)育了更高的花數(shù)和葉數(shù)，棉花增加了纖維生物量。干旱脅迫試驗(yàn)表明，缺水條件下，在成熟期長春花植株中引入MWCNTs可提高植物存活率，且無葉片枯萎癥狀[36]。賀君杰等發(fā)現(xiàn)5 mg/L的MWCNTs可以通過調(diào)節(jié)重金屬轉(zhuǎn)運(yùn)ATP酶5（ATPase5）基因的表達(dá)減少銅離子吸收、提高根部非蛋白硫醇的水平以螯合過量的銅離子等方式緩解苜蓿銅脅迫[43]。

CNTs在作物抗逆中的作用既有植物生理作用也有基因表達(dá)調(diào)控作用，機(jī)理較復(fù)雜。

表1 作物培育研究中的CNTs參數(shù)

注：SWCNT：單壁碳納米管；MWCNT：多壁碳納米管。

Note: SWCNT: Single-walled carbon nanotube; MWCNT: Multi-walled carbon nanotube.

2.3 碳納米管在作物監(jiān)測中的應(yīng)用

核酸適配體、蛋白質(zhì)或特殊ssDNA片段修飾的SWCNTs具有很好的近紅外熒光特性，已經(jīng)被廣泛用來研制各種生物傳感器[44]，基于熒光強(qiáng)度變化的傳感器工作原理如圖2a所示，當(dāng)SWCNTs周圍的化學(xué)環(huán)境發(fā)生變化時(shí)，其熒光強(qiáng)度或熒光中心波長會(huì)發(fā)生變化。例如，Lambert等利用ssDNA纏繞的SWCNTs高選擇性地檢測玉米黃曲霉毒素B1（aflatoxin B1，AFB1），而不受伏馬菌素（fumonisin B1，F(xiàn)B1）等毒素的影響[45]，另外，基于CNTs的電化學(xué)傳感器也可以實(shí)現(xiàn)對(duì)真菌毒素的檢測[46]。Wu等報(bào)道了利用對(duì)H2O2敏感的DNA適配體修飾的SWCNTs來檢測H2O2，實(shí)現(xiàn)通過SWCNTs熒光的強(qiáng)弱變化實(shí)時(shí)反映植物體內(nèi)H2O2的含量變化，原理如圖 2b所示[47]，有望實(shí)現(xiàn)對(duì)脅迫下植物體內(nèi)ROS變化（時(shí)間點(diǎn)和位置）的監(jiān)測。Lew等利用ssDNA修飾的SWCNTs實(shí)現(xiàn)了對(duì)萵苣、芥菜、菠菜、大頭菜、酸模和擬南芥中創(chuàng)傷葉片處H2O2含量的實(shí)時(shí)在線測量[48]。

雖然SWCNTs表面可以較為容易地修飾多種生物大分子，但是修飾過程會(huì)改變適配體或DNA的構(gòu)象，這可能會(huì)影響其與靶標(biāo)分子的特異性結(jié)合，而這種結(jié)合是否具有可逆性的熒光響應(yīng)也是需要解決的問題。

注：當(dāng)基于熒光響應(yīng)的CNTs傳感器與待測物結(jié)合后，其熒光的光強(qiáng)或中心波長會(huì)發(fā)生變化。δI為光強(qiáng)的變化量，δλ為中心波長的變化量。

2.4 碳納米管在DNA/RNA遞送中的應(yīng)用

SWCNTs的直徑很小，表面修飾DNA后的復(fù)合物直徑仍然小于植物細(xì)胞壁孔隙尺寸（例如，原始SWCNTs直徑1.3 nm，聚合物修飾后的直徑為8.1 nm，再經(jīng)DNA培育后的直徑變?yōu)?6.3 nm[49]），能夠穿透植物細(xì)胞壁[50]。不過，SWCNTs的長度可能會(huì)影響其穿過細(xì)胞壁，因此一般需要將SWCNTs變短。

與傳統(tǒng)植物DNA遞送和小干擾RNA（small interfering RNA，siRNA）施用方法不同，CNTs介導(dǎo)的DNA/RNA遞送具有瞬時(shí)轉(zhuǎn)化率高的特點(diǎn)[51-52]，而且CNTs還可以保護(hù)多核苷酸避免被核酸酶降解[53]。目前，SWCNTs被認(rèn)為是基因遞送的理想載體[54-55]，例如：Kwak等利用殼聚糖修飾的SWCNTs選擇性地將質(zhì)粒DNA遞送至葉綠體，在成熟的芥菜、金蓮花、煙草和菠菜植物以及分離的擬南芥葉肉原生質(zhì)體中實(shí)現(xiàn)了葉綠體靶向轉(zhuǎn)基因遞送和瞬時(shí)表達(dá)，轉(zhuǎn)化率達(dá)到88%[56]。Demirer等通過共價(jià)修飾和非共價(jià)修飾的SWCNTs實(shí)現(xiàn)了在本氏煙草、芝麻菜、小麥、棉葉和芝麻菜原生質(zhì)體上的高效DNA遞送和強(qiáng)蛋白表達(dá)[49,57]。

Apartsin等詳述了siRNA綁定CNTs形成復(fù)合物的方法、靶細(xì)胞如何吸收siRNA-CNTs復(fù)合物以及細(xì)胞內(nèi)siRNA如何解綁等[58]。Edwards等將雙鏈DNA（double-stranded DNA，dsDNA）連接到聚酰胺-胺聚合物（PAMAM）修飾的MWCNTs上形成的PAMAM- CNT- dsRNA復(fù)合物注射到赤擬谷盜幼蟲體內(nèi)時(shí)，發(fā)現(xiàn)PAMAM-CNT在細(xì)胞液泡和細(xì)胞核中可見，而且兩個(gè)靶基因α-微管蛋白和線粒體RNA聚合酶的敲除水平顯著增加。與單獨(dú)注射dsRNA（靶基因α-微管蛋白）相比，注射PAMAM-CNT-dsRNA的幼蟲發(fā)現(xiàn)皰翅表型的發(fā)生率更多，這表明，使用功能化的CNTs進(jìn)行dsRNA遞送可以提高RNA干擾對(duì)害蟲物種的功效[59]。

碳納米管用于DNA/RNA遞送的關(guān)鍵是基因的“裝載”。碳納米管的結(jié)構(gòu)決定了dsRNA和dsRNA很難直接纏繞到CNTs上[60]，文獻(xiàn)中主要有兩種形式實(shí)現(xiàn)dsDNA/dsRNA遞送：1）先將兩條互補(bǔ)的單鏈DNA（single-stranded DNA，ssDNA）/ssRNA分別纏繞到CNTs表面，當(dāng)上述混合物進(jìn)入細(xì)胞后，ssDNA或ssRNA與CNTs解吸附，再形成dsDNA或dsRNA，原理如圖3a所示[50]；2）先使用聚合物修飾CNTs，然后通過電荷吸附作用或形成絡(luò)合物等方式將dsDNA或dsRNA固定在CNTs表面，原理如圖3b所示[59]。另外，還有一種方法可以嘗試：原理如圖3c所示[61-62]，將dsDNA或dsRNA與一段ssDNA相連，ssDNA通過π-π堆積纏繞到CNTs表面后，即可將dsDNA或dsRNA固定在CNTs上。

圖3 DNA/RNA遞送的操作方法

3 作物研究中碳納米管的使用問題分析

3.1 碳納米管培養(yǎng)基的有效濃度問題

濃度是研究CNTs影響作物生長發(fā)育必須衡量的參數(shù)，雖然文獻(xiàn)中都給出了所使用的CNTs培養(yǎng)基濃度，但實(shí)際能夠進(jìn)入植物細(xì)胞的有效CNTs濃度是不確定的[63]。這是因?yàn)橐徊糠治墨I(xiàn)中采用超聲直接分散的原始未修飾的CNTs，分散效果較差，CNTs聚集團(tuán)較多（如圖4a所示[16]，箭頭處為聚集團(tuán)，Copyright 2009 American Chemical Society）；另一部分文獻(xiàn)采用超聲分散的表面羧基化的CNTs，由于羧基與CNTs的質(zhì)量比和體積比均過低，其在一定時(shí)間后仍然會(huì)產(chǎn)生聚集、沉淀。

盡管文獻(xiàn)中的透射電子顯微鏡或熒光圖像顯示植物細(xì)胞中的CNTs往往是小的聚集團(tuán)[64]（如圖4b所示[65]，Copyright 2021 American Chemical Society），尺寸在數(shù)μm甚至百μm量級(jí)[53]，但目前還沒有直接證據(jù)證明CNTs是以聚集團(tuán)的形式進(jìn)入的細(xì)胞。與之相反的是，Husen等論證了當(dāng)CNTs通過毛細(xì)作用被輸送到通道狹窄的點(diǎn)時(shí)，CNTs會(huì)積聚并阻塞營養(yǎng)物質(zhì)進(jìn)一步流動(dòng)的通道[66]；Martínez等使用超聲分散的未修飾MWCNTs-霍格蘭溶液（直徑6～9 nm，長度0.1～0.5 μm）水培西蘭花，發(fā)現(xiàn)在植株根部和莖部的CNTs都是以單根的形式存在[30]。綜上分析，CNTs應(yīng)該是以單根形式穿過細(xì)胞壁，并會(huì)在細(xì)胞質(zhì)中的聚集，而CNTs培養(yǎng)基中的聚集團(tuán)是不太可能整體進(jìn)入植物細(xì)胞的。因此，研究者應(yīng)當(dāng)注意測量CNTs的有效濃度。

圖4 試驗(yàn)中CNTs的典型形態(tài)

3.2 碳納米管的特異性作用問題

試驗(yàn)中可采用的CNTs商品較少，只有表面氨基化、羧基化或羥基化的幾類。由于并未在表面修飾特殊功能的材料，因此在研究作物植株受CNTs影響時(shí)，CNTs往往是被當(dāng)作一種納米尺寸的材料使用，試驗(yàn)結(jié)果也與大部分的NPs（如CeO2、TiO2、ZnO等）對(duì)作物培育的影響相同：都可以對(duì)種子引發(fā)、種子萌發(fā)、幼苗生長過程、對(duì)抗脅迫壓力產(chǎn)生有益影響，從而提高種子發(fā)芽率，增大芽長和根長[18,67-69]。究其原因，基本共識(shí)是由于NPs可以導(dǎo)致細(xì)胞壁穿孔，增強(qiáng)了水的吸收、改變了種子的抗氧化系統(tǒng)等[19]，亦即在促進(jìn)作物生長方面，與其他NPs相比，無修飾或非選擇性修飾的CNTs并未顯示具有獨(dú)特的作用效果。

3.3 碳納米管帶來的食品安全問題

無論是被用于人類/動(dòng)物疾病治療、植入式傳感，還是被用于農(nóng)業(yè)，CNTs的生物毒性一直是被討論的問題。相比于人類/動(dòng)物領(lǐng)域主要研究CNTs的直接毒性，農(nóng)業(yè)中還需要考慮其間接毒性問題[70]。

3.3.1 碳納米管的直接毒性

美國國家職業(yè)安全衛(wèi)生研究所在職業(yè)性癌癥致癌物清單中并未將CNTs列為致癌物[71]，但其一項(xiàng)研究表明，接觸CNTs的工人離體全血樣本對(duì)兩種微生物刺激劑（脂多糖和葡萄球菌腸毒素）功能性免疫反應(yīng)較低[72]。

REZAEI等創(chuàng)建了用于全面調(diào)查番茄植株器官中積累的MWCNTs可能帶來的健康風(fēng)險(xiǎn)評(píng)估平臺(tái)，用CNTs污染植株結(jié)出的西紅柿喂養(yǎng)小鼠，結(jié)果顯示所有參與評(píng)估的動(dòng)物器官和生理參數(shù)都沒有顯示出毒性反應(yīng)，表明由于根部施用導(dǎo)致在植物不同部位積累的CNTs數(shù)量不足以對(duì)小鼠產(chǎn)生毒性[73]。

3.3.2 碳納米管的間接毒性

CNTs中的碳原子主要以sp2雜化形式存在，管壁碳原子的P電子形成大范圍的未配對(duì)的離域π電子云，因此可以通過π-π堆積（π-stacking）與其他含有碳環(huán)狀結(jié)構(gòu)的物質(zhì)（如芳香族化合物）結(jié)合[74-77]。另外，表面修飾物也會(huì)與離子結(jié)合，如吸附水中重金屬離子（鎘、稀土、鉛、銅離子等）[78-80]。上述吸附作用或離子結(jié)合作用使得CNTs可以作為載體將外源污染物運(yùn)輸?shù)睫r(nóng)作物的多個(gè)部位，造成外源有毒物質(zhì)在植株內(nèi)的積累，例如：

1）Chen等研究發(fā)現(xiàn)，在超聲分散的MWCNTs與污染物（六氯苯HCB、滴滴涕p-p′ DDT）的同時(shí)作用下，污染物在芥菜植株內(nèi)的富集，并給出了幾種污染物的動(dòng)力學(xué)積累過程[81]。WILD等發(fā)現(xiàn)超聲分散MWCNTs穿透過的小麥根部細(xì)胞壁更加容易使大分子菲進(jìn)入該細(xì)胞[16]，但是沒說明菲是由于π-π堆積作用附著在碳管壁一起進(jìn)入的還是由被碳管擴(kuò)大的細(xì)胞壁孔洞進(jìn)入的細(xì)胞。

2）Wang等的研究表明，羧化的MWCNTs與Pb+Cd的組合導(dǎo)致蠶豆幼苗營養(yǎng)元素不平衡，葉片中Pb和Cd的富集引起葉片氧化脅迫和損傷，而單獨(dú)的MWCNTs-COOH或Pb+Cd作用卻沒有上述效果[82]。

相較而言，含有CNTs的作物食用安全問題，可以直接參考動(dòng)物或人類直接CNTs暴露的毒性研究結(jié)論。但是，在含有污染物的植株生長環(huán)境中，CNTs運(yùn)載外源毒性物質(zhì)進(jìn)入植株帶來的間接毒性問題需要更深入的研究。

4 作物研究中碳納米管試驗(yàn)方案建議

4.1 碳納米管選型建議

SWCNTs與MWCNTs具有同類型的特性，都具有很高的比表面積和長徑比、高的化學(xué)反應(yīng)活性等[83-84]，但SWCNT在幾何尺寸、熒光特性和生物毒性方面具有明顯優(yōu)勢。通過綜合分析文獻(xiàn)可知：

1）大直徑的MWCNTs對(duì)作物培育的負(fù)面影響較少。

2）考慮到細(xì)胞壁的通過性，直徑越小的CNT越容易穿過細(xì)胞壁，或允許“裝載”更大的分子。

3）CNT是一維納米材料，長度也會(huì)影響植物細(xì)胞的吸收。文獻(xiàn)中尺寸短的CNTs對(duì)作物的影響更顯著。

4）雖然有文獻(xiàn)指出共價(jià)修飾后的MWCNTs可以產(chǎn)生熒光（激發(fā)光中心波長350 nm，熒光中心波長為418 nm）[85-86]，但其不是常用的熒光物質(zhì)。而非共價(jià)修飾的SWCNTs具有非常優(yōu)秀的熒光特性[87]，其典型手性CNTs的熒光發(fā)射峰在近紅外Ⅱ區(qū)，遠(yuǎn)離植物色素的自發(fā)熒光波段，避免植物組織自發(fā)熒光的干擾，在表征方面具有優(yōu)勢。

5）世界衛(wèi)生組織國際癌癥研究機(jī)構(gòu)公布的致癌物清單中，MWCNTs被認(rèn)定為2B類致癌物，而SWCNT被認(rèn)定為3類致癌物[88]。SWCNT的直接生物毒性比MWCNT小很多。

因此，本文建議在作物培育的研究中使用短型大直徑多壁碳納米管作為試驗(yàn)材料；而在以遞送和傳感為主的作物研究中，使用短型單壁碳納米管作為試驗(yàn)材料。

4.2 碳納米管培養(yǎng)基制備方法建議

由于碳納米管疏水性強(qiáng)、易聚集，若采用超聲分散未修飾的或表面羧基化的CNTs來制備溶液培養(yǎng)基，或采用將CNTs粉末與瓊脂直接攪拌混合的方法制備瓊脂培養(yǎng)基[34,89]，不同的研究者很難獲得相同的有效濃度。

為了保證濃度對(duì)作物影響結(jié)果的可復(fù)現(xiàn)性，本文建議采用表面修飾的碳納米管進(jìn)行試驗(yàn)研究，修飾物可以為在植株體內(nèi)可降解的有機(jī)分子如ssDNA、吡唑醚菌酯等[90-91]，由于營養(yǎng)液中的陽離子可能與CNTs表面修飾物發(fā)生反應(yīng)，因此不建議用營養(yǎng)液來分散CNTs。

CNTs培養(yǎng)基儲(chǔ)備液的制備流程建議為CNTs和修飾物的水溶液混合后在水浴杯中進(jìn)行超聲分散，將分散液高速離心4 h后取80%～90%的上層溶液，利用分光光度計(jì)測量CNTs濃度。

由于制備瓊脂時(shí)需要將水加熱至接近沸騰，而修飾物在高溫下，構(gòu)象可能會(huì)發(fā)生改變甚至解析附，故制備CNTs瓊脂培養(yǎng)基的流程建議為瓊脂粉與少量高純水混合后加熱至全部溶解，再加入CNTs儲(chǔ)備液攪拌均勻并定容。

基于上述兩種培養(yǎng)基，可以方便地利用水培法和瓊脂培育法探索CNTs在作物研究中的各種作用[30]。

4.3 植株內(nèi)CNTs的表征方法

一般采用透射電子顯微鏡和掃描電鏡對(duì)CNTs的形貌[92]或植物組織切片成像[93]。采用電子斷層掃描（electron tomography，ET）可以獲得細(xì)胞的3D圖像，在觀察CNTs在植物細(xì)胞內(nèi)的體分布方面具有更大的優(yōu)勢[94]。

熒光顯微鏡也常用于表征CNTs?？紤]到顯微鏡細(xì)胞成像系統(tǒng)的通用性，一般農(nóng)學(xué)生物實(shí)驗(yàn)室常采用以下3種用法：1）采用對(duì)細(xì)胞染色的方法對(duì)細(xì)胞成像，利用CNTs在該激發(fā)光波段（如370、488、592 nm等）無熒光的特性，通過研究圖像中黑斑來表示其形態(tài)和在細(xì)胞中的位置[64]；2）將熒光團(tuán)修飾到CNTs表面進(jìn)行熒光標(biāo)記，而不對(duì)細(xì)胞染色，通過融合熒光圖像與細(xì)胞的明場圖像來研究其相對(duì)位置（如圖5a所示[95]，Copyright 2009 American Chemical Society）；3）將CNTs作為熒光團(tuán)的淬滅物質(zhì)：進(jìn)入細(xì)胞前，連接在探針兩端的熒光團(tuán)和CNTs之間會(huì)緊密接觸，使得熒光被淬滅。進(jìn)入細(xì)胞后，當(dāng)探針與目標(biāo)物結(jié)合后，由于構(gòu)象發(fā)生變化，熒光團(tuán)脫離CNTs表面并發(fā)射熒光（如圖5b所示[53]）。

圖5 植株內(nèi)CNTs的不同表征方式

另外，SWCNTs具有非常優(yōu)秀的熒光特性，其激發(fā)光范圍為550～800 nm，熒光范圍為900～1 400 nm[96]（如圖5c所示）。近紅外熒光顯微鏡可以被用來檢測SWCNTs的熒光，圖像包含熒光光強(qiáng)和二維空間位置信息[97]，近紅外共聚焦熒光顯微鏡景深小，可以用于確定SWCNTs在細(xì)胞內(nèi)的深度[98]。雙光子激發(fā)熒光顯微鏡（two-photon excitation microscopy，TPEM）更適用于檢測MWCNTs的熒光（360～530 nm），因?yàn)榧ぐl(fā)光710 nm恰好在植物組織的近紅外窗口區(qū)，與350 nm的激發(fā)光相比優(yōu)勢明顯。同時(shí)，基于多幅TPEM圖像的3D重建技術(shù)，可以更清晰地分辨MWCNTs與植物細(xì)胞的位置關(guān)系（如圖5d所示[16]，Copyright 2009 American Chemical Society）。

5 結(jié)論與展望

碳納米管在農(nóng)作物生長發(fā)育、抗逆、育種等方面的積極作用已經(jīng)被證實(shí)，應(yīng)用前景廣闊。但也不可避免地存在需要改進(jìn)的情況，例如，未修飾的和共價(jià)修飾的CNTs對(duì)作物培育的影響已經(jīng)有豐富的研究基礎(chǔ)，但非共價(jià)修飾的CNTs的應(yīng)用研究相對(duì)較少。因此，對(duì)CNTs在作物研究中的應(yīng)用總結(jié)與展望如下：

1）CNTs穿透細(xì)胞壁的原理、進(jìn)入細(xì)胞后的聚集機(jī)制及其在細(xì)胞間轉(zhuǎn)運(yùn)的動(dòng)力學(xué)原理尚缺乏透徹研究，需要設(shè)計(jì)新穎的試驗(yàn)、采取新的表征手段或采用新的植入式在線傳感器進(jìn)行研究。

2）基于CNTs的植入式在線監(jiān)測傳感器將是CNTs在作物研究中的重要應(yīng)用方向之一。相對(duì)于被用作體外傳感器，由于CNTs可以穿透細(xì)胞壁，具有優(yōu)良的近紅外熒光特性，適當(dāng)濃度的CNTs對(duì)作物沒有生物毒性（甚至有益于作物的生長），因此，非共價(jià)修飾的CNTs更適合作為植入式傳感器，對(duì)植物體內(nèi)的多種化學(xué)物質(zhì)進(jìn)行實(shí)時(shí)在線監(jiān)測。

3）作為轉(zhuǎn)運(yùn)載體是CNTs在作物研究中的一個(gè)重要應(yīng)用方向：在siRNA遞送方面，siRNA-CNTs復(fù)合物在施用周期、植物細(xì)胞吸收、害蟲細(xì)胞吸收、保護(hù)RNA被降解方面均有獨(dú)特的優(yōu)勢，但是，相關(guān)文獻(xiàn)并沒有進(jìn)行包括siRNA-CNTs復(fù)合物的施用、植物細(xì)胞吸收、害蟲細(xì)胞吸收、阻斷基因表達(dá)、害蟲發(fā)育停滯或者死亡這一系列完整的試驗(yàn)流程，因此，CNTs介導(dǎo)的RNA干擾抗蟲技術(shù)還有廣闊的研究空間。

4）利用CNTs對(duì)有機(jī)農(nóng)藥的吸附，實(shí)現(xiàn)殺菌劑的精準(zhǔn)定點(diǎn)運(yùn)載及農(nóng)藥緩釋，用來提高農(nóng)藥的藥效以及延長半衰期也是一個(gè)重要的研究方向。

另外，CNTs與動(dòng)物相互作用（遞送、檢測、疾病治療）的研究深度和廣度遠(yuǎn)超其與植物的相互作用研究。近年來，已經(jīng)有將動(dòng)物研究方面的技術(shù)應(yīng)用轉(zhuǎn)化到植物方面的例子。在未來，將CNTs在動(dòng)物應(yīng)用研究中的經(jīng)驗(yàn)轉(zhuǎn)化到植物應(yīng)用研究中將會(huì)有效地促進(jìn)農(nóng)作物培育的發(fā)展。

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Progress and analysis of carbon nanotube applications in crop research

LIU Xuewen1, GAO Juntao2, LI He1, YANG Zishang1, CHEN Jing1※

(1.,,,450002,2.,,450047,)

The promotion and biotoxic effects of carbon nanotubes (CNTs) on crop cultivation have drawn much attention over the last decade. The studied species have covered common crops, such as rice, corn, wheat, tomato, and grapes. There was great inconsistency in the previous experiments, including the various species, growth stages, and cultivation methods of crops as well as the various types, dispersion, application, and concentrations of CNTs, resulting in the diversification of test results in the literature. To establish a relatively unified experiment framework for carbon nanotubes, this study first reviewed the progress of the application of carbon nanotubes in following four crop research directions: crop cultivation, stress resistance, physiological index monitoring, and agricultural genetic engineering, and summarized the carbon nanotube parameters used in the literature and their application effects. It was found that the ultrasonic method or carboxylation covalent modification was commonly used to disperse the CNTs for crop cultivation research, which would result that the effective concentration of carbon nanotubes in crop research being unknown. Meanwhile, this kind of non-selectively modified CNTs was used as the type of nano-sized material. There was no significant difference in the effects between NPs and CNTs on the crops. It is still lacking in the unique effects of CNTs on crop growth. Then, the non-covalently modified CNTs were proposed to establish the unique role of CNTs in crop cultivation, for example, they can aggregate at the target sites after CNTs were modified by macromolecules that can be decomposed by special plant enzymes. Biomolecules (e.g., proteins, and nucleic acids), and polymers (e.g., chitosan, PEG, PEI, and PAMAM) can non-covalently be modified on the surface of CNTs, and further used to adsorb the molecules with negative charges, which can be used as carrier or sorbent. In addition, the non-covalent modification caused no defects on the CNT surface, indicating the fluorescence properties of SWCNTs were retained. So, CNTs were often used for DNA/RNA delivery and the development of biosensors. The working principles and application scenarios of selectively modified CNTs were also summarized as: 1) The CNTs were undoubtedly an important research direction for the applications as the carriers of gene transfer. However, the DNAs that were carried by CNTs were difficult to achieve stable gene transformation, in terms of transgenic applications. By contrast, the siRNA-CNTs complexes shared unique advantages, in terms of administration cycle, plant cell uptake, and protection of RNA from degradation during non-transformative gene delivery. One of the future development directions was in the field of agricultural non-transgenic insect-resistant. 2) The surface of SWCNTs was non-covalently modified with a variety of organic substances. The target molecules were then bound to the organic substances. A variety of sensors were obtained using their fluorescence or electrochemical properties. Some of them were more suitable as implantable sensors for the real-time online monitoring of multiple chemicals in plants, compared with the in vitro sensors. The reason was that the CNTs were used to penetrate the cell walls, indicating excellent near-infrared fluorescence properties. There was no biotoxic effect on the crops at appropriate concentrations (and even beneficial to crop growth). Furthermore, the toxicity problem was caused by the accumulation of exogenous toxic substances in the plant, due to the adsorption or ion exchange with the modified CNTs. It was more worthy of investigation than the toxicity problem of CNT itself. Finally, suggestions for the CNTs experiment program in crop research are given, including the selection of carbon nanotubes, the preparation of medium and characterization methods. To sum up, unlike the previous crop cultivation using pristine and covalently modified CNTs, in the future, using non-covalently modified CNTs or non-covalent stacking of CNTs for crop research is an important development direction.

nanometer; crops; carbon nanotube; surface modification; crop cultivation; biotoxicity

2022-12-29

2023-03-02

河南省教育廳國際科技合作項(xiàng)目（222102520017）；財(cái)政部和農(nóng)業(yè)農(nóng)村部：國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系項(xiàng)目（CARS-04）

劉學(xué)文，博士，高級(jí)工程師，研究方向?yàn)橹悄芨兄夹g(shù)。Email：liuxuewen1983@hotmail.com

陳靜，博士，碩士生導(dǎo)師，研究方向?yàn)樯飩鞲衅鳌⒐怆娦畔⑻幚砑夹g(shù)。Email：jingchen1983@outlook.com

10.11975/j.issn.1002-6819.202212182

S-1

A

1002-6819(2023)-08-0001-11

劉學(xué)文，高軍濤，李赫，等. 碳納米管在作物研究中的應(yīng)用進(jìn)展與分析[J]. 農(nóng)業(yè)工程學(xué)報(bào)，2023，39(8)：1-11. doi：10.11975/j.issn.1002-6819.202212182 http://www.tcsae.org

LIU Xuewen, GAO Juntao, LI He, et al. Progress and analysis of carbon nanotube applications in crop research[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2023, 39(8): 1-11. (in Chinese with English abstract) doi：10.11975/j.issn.1002-6819.202212182 http://www.tcsae.org