王圣瓊, 孫 暢, 陶春先, 韓朝霞, 洪瑞金, 林 輝, 張大偉

(1.上海理工大學(xué) 光電信息與計算機工程學(xué)院, 上海 200093; 2.復(fù)旦大學(xué) 高分子科學(xué)系, 上海 200433;3.上海理工大學(xué) 教育部光學(xué)儀器與系統(tǒng)工程研究中心, 上海 200093;4.上海理工大學(xué) 上海市現(xiàn)代光學(xué)系統(tǒng)重點實驗室, 上海 200093)

引 言

以金、銀為代表的亞納米尺度貴金屬發(fā)光結(jié)構(gòu)由于其良好的物理化學(xué)穩(wěn)定性、較高的熒光量子產(chǎn)額、在一定條件下的開/關(guān)可控發(fā)光等特性,使得其在生物熒光探針[1]、化學(xué)傳感器[2- 3]、光存儲/光編碼[4- 5]、無稀土熒光粉[6]等領(lǐng)域顯示出了很好的應(yīng)用前景。由于亞納米尺度金、銀的尺寸非常小(一般<2 nm),通常需要基質(zhì)材料加以穩(wěn)定以防止其團聚,常見的基質(zhì)材料有硫醇[7- 9]、半胱氨酸[10- 11]、DNA[12]、蛋白質(zhì)[13]、聚合物[14- 16]等,對于無機基質(zhì)材料,目前有見報道的只有沸石[17- 22]和玻璃[23- 24]兩種。

在眾多有機配體材料中,牛血清蛋白由于具有優(yōu)異的生物兼容性、水溶性、低廉的價格,以及合成獲得的牛血清蛋白-Au配合物(BSA-Au)樣品具有較高的發(fā)光強度而被廣泛研究[25]。

以往,多數(shù)研究者認為BSA-Au樣品的發(fā)光機理是基于金屬自由電子理論和久堡模型,即當(dāng)金的尺寸減小到與其費米波長相當(dāng)時,其原本準(zhǔn)連續(xù)的電子能級會發(fā)生劈裂,當(dāng)最高占據(jù)分子軌道(highest occupied molecular orbital,HOMO)與最低未占據(jù)分子軌道(lowest unoccupied molecular orbital,LUMO)之間的能隙劈裂到可以產(chǎn)生輻射發(fā)光電子躍遷時,對應(yīng)金的臨界尺寸約為2 nm[26]。最近,我們的實驗結(jié)果表明,BSA-Au樣品的寬帶紅光發(fā)射很可能不是起源于量子尺寸限制效應(yīng),而很可能是來自由配體到Au(I)離子的電荷轉(zhuǎn)移發(fā)光[27]。電荷轉(zhuǎn)移發(fā)光通常具有較大的斯托克斯位移,對于BSA-Au樣品,其最強的激發(fā)峰位于紫外區(qū)域,而紫外光對細胞有殺傷力,對人體組織有一定危害。因此,增強BSA-Au樣品在長波區(qū)域的激發(fā)效率有益于實際應(yīng)用?；诰钟虮砻娴入x激元效應(yīng),通過調(diào)節(jié)金納米顆粒的尺寸、形狀等參數(shù),可以調(diào)控其在可見光到近紅外波段的吸收性能。而將具有適當(dāng)吸收的金納米顆粒與BSA-Au樣品進行復(fù)合,預(yù)期能夠增強BSA-Au樣品在長波區(qū)域的光吸收,同時局域表面等離激元共振效應(yīng)也可能對BSA-Au的熒光強度有所增強。

本文采用一鍋合成法制備了BSA-Au樣品,將其與進行過表面修飾而帶有正電荷的Au納米顆粒通過庫侖力作用形成復(fù)合物,測試和分析了復(fù)合物樣品的吸收和熒光光譜,并對熒光光譜變化進行了分析討論。

1 原 理

由于BSA分子中存在的巰基與重金屬離子(如Au(Ⅰ)離子)間存在很強的相互作用,因此可以合成出BSA-Au配合物。而對于BSA-Au配合物,因其在可見光區(qū)域的吸收相比其紫外光區(qū)域的吸收較弱,本文通過庫侖力作用將帶負電的BSA-Au與表面帶有正電荷的金納米顆粒進行復(fù)合,并通過金納米顆粒的局域表面等離激元共振來增強BSA-Au配合物在可見光區(qū)域的吸收。

2 樣品制備及測試

所有化學(xué)試劑均采購自日本和光化學(xué)試劑有限公司,主要采用氯金酸(HAuCl4·H2O)、牛血清蛋白(BSA)、氫氧化鈉(NaOH)等試劑合成BSA-Au樣品。

2.1 樣品制備

(1) BSA-Au(Ⅰ)樣品的制備

采用文獻[25]中報道一鍋法制備BSA-Au樣品。合成后的樣品溶液采用8～14 kDa((1 Da=1 u=(1.660 540 2±0.000 001 0)×10-27kg)的半透膜對去離子水進行滲析,每隔4 h更換1次去離子水(DI水),以去除在溶液中游離的Na+離子、OH-離子、Au(Ⅰ)離子、Au(Ⅲ)離子和蛋白質(zhì)小碎片等雜質(zhì)[28],直至BSA-Au(Ⅰ)溶液的pH到達7時停止?jié)B析。

(2) Au納米顆粒@BSA-Au(Ⅰ)復(fù)合物樣品的制備

將500 μL和2.5 mL表面帶有正電荷的金納米顆粒(約1×1016個/mL)溶液分別緩慢滴入2份攪拌中的500 μL的BSA-Au水溶液中,這樣帶有負電荷的BSA-Au樣品將會與表面帶有正電荷的金納米顆粒通過庫侖力作用結(jié)合到一起,得到復(fù)合物樣品,記為Au NP@BSA-Au,分別簡記為C1和C2。

2.2 測試表征

采用基質(zhì)輔助激光解析電離-飛行時間質(zhì)譜儀(MALDI-TOF MS)測試了BSA-Au樣品的金含量。采用紫外-可見分光光度計測試了樣品的吸收光譜,測試范圍為300～800 nm。采用Horiba Fluorolog 3型熒光光譜儀測試了樣品的熒光光譜。

3 結(jié)果與討論

圖1是純BSA與BSA-Au樣品的MALDI-TOF MS圖譜,從圖中可以看出:純BSA對應(yīng)的質(zhì)荷比約為66 500;而對于BSA-Au樣品,樣品的信號峰出現(xiàn)了寬化,且其峰值對應(yīng)的質(zhì)荷比減小至約65 900,這是由于BSA-Au合成過程中經(jīng)歷了強酸和強堿環(huán)境,導(dǎo)致BSA分子以小碎片形式損失掉一部分質(zhì)量所造成的。由此可見,通過MALDI-TOF MS精確分析BSA-Au樣品中金的精確含量存在一定的困難。

圖1 純BSA與BSA-Au樣品的MALDI-TOF MS圖譜Fig.1 MALDI- TOF MS measurement for the pure BSA and BSA- Au sample

圖2是BSA-Au樣品以及Au NP@BSA-Au樣品(C1和C2)的吸收光譜。圖2(a)中BSA-Au樣品在可見光波段并無顯著吸收峰,隨著波長的減小,吸收逐漸增強;對于Au NP@BSA-Au樣品,位于536 nm和538 nm的吸收峰對應(yīng)于金納米顆粒的局域表面等離激元共振吸收。而在圖2(b)的熒光光譜中,Au NP@BSA-Au復(fù)合物的熒光強度相比BSA-Au(I)樣品大幅減弱,產(chǎn)生了嚴(yán)重的熒光猝滅現(xiàn)象。

導(dǎo)致BSA-Au樣品產(chǎn)生熒光猝滅的原因可能是:在BSA-Au與金納米顆粒結(jié)合過程中,正負電荷抵消掉一部分,而前期的研究結(jié)果表明,樣品所攜帶負電荷的數(shù)量與BSA-Au樣品紅光熒光強度正相關(guān)[27],因此導(dǎo)致了熒光猝滅。

圖3為BSA-Au樣品以及Au NP@BSA-Au樣品(C1和C2)的激發(fā)-發(fā)射3D等高圖。可以看出,Au NP@BSA-Au樣品的最強激發(fā)峰仍位于紫外區(qū)域(約375 nm處),即雖然金納米顆粒通過表面等離激元共振增強了Au NP@BSA-Au樣品在可見光區(qū)域的光吸收,但并沒有有效增強BSA-Au在可見光區(qū)域的激發(fā)效率。

圖2 BSA-Au,Au NP@BSA-Au樣品的吸收光譜和熒光光譜Fig.2 Optical absorption and photoluminescence spectra of the BSA- Au and Au NP@BSA- Au samples

圖3 不同樣品的激發(fā)-發(fā)射3D等高圖Fig.3 Excitation- emission 3D contours of different samples

4 結(jié) 論

制備了BSA-Au和BSA-Au/金納米顆粒復(fù)合物樣品,采用MALDI-TOF MS分析了BSA-Au樣品中的金含量,并分析了研究中存在的困難。測試了樣品的吸收光譜和熒光光譜。實驗結(jié)果表明,在本文工作實驗條件下,金納米顆粒雖然增強了復(fù)合物樣品在可見光區(qū)域的光吸收,但對BSA-Au紅光發(fā)光有猝滅作用,后續(xù)工作需要改進復(fù)合物的結(jié)構(gòu)設(shè)計。

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