發展可偵測胰島素之官能化奈米粒子親和質譜法

Abstract

胰島素可以調控人體中碳水化合物和脂肪的代謝，並且是一可控制血糖濃度的賀爾蒙。因此，血液中胰島素的濃度已被用於診斷疾病如糖尿病等的指標。開發快速和可靠的分析方法用於檢測血清中胰島素含量應有一定的重要性。我們最近已經利用一鍋反應合成出表面修飾有右旋糖酐的金奈米團簇，而在本論文中我們發現此金奈米團簇對於胰島素具有特殊的辨識能力。為了便於分離，我們將此金奈米團簇修飾在四氧化三鐵磁性奈米粒子表面上，發展用為可濃縮萃取胰島素的親和奈米磁性探針。胰島素是由以雙硫鍵結合的A鏈和B鏈所組成的，此雙硫鍵可利用還原法切斷而可以得到個別的A鏈和B鏈。當利用由金奈米團簇-四氧化三鐵磁性奈米粒子的官能化奈米探針進行含A鏈和B鏈的樣品之選擇性濃縮實驗，再利用基質輔助雷射脫附游離質譜法進行分析結果確認，實驗結果發現此官能化探針可選擇性地抓取胰島素B鏈。為了進一步確認分析結果，實驗中再使用胰島素的胰蛋白酶消化產物為樣品，而以相同官能化奈米探針進行此樣品之濃縮萃取並藉由基質輔助雷射脫附游離質譜法進行分析，結果顯示，僅有來自於B鏈的胜肽片段(＃1-22)出現在質譜圖中，表示該胜肽片段是胰島素與此官能化奈米探針的結合位置。此外，也將該肽序列衍生出的三條合成胜肽進一步用為官能化探針的濃縮萃取樣品，並利用基質輔助雷射脫附游離質譜法確認結果，結果顯示僅有來自於代表B鏈胜肽序列(＃8-18)的離子峰出現在質譜圖中，表示該胜肽片段是胰島素與官能化奈米探針的結合序列。除此之外，我們的實驗結果也顯示在此所發展的方法對於胰島素的偵測極限可低於〜1 pM左右。由於血液中胰島素的濃度通常介於幾十到幾百pM之間，所以我們所發展的方法應該可以適用於真實樣品如血清中胰島素的分析。在本論文的最後，也探討了對血清中胰島素的定量分析之可行性，所得到的誤差範圍在~16% 左右。因此，我們所發展的方法應該有用於實際樣品分析的潛力。

Insulin is a hormone that can regulate the metabolism of carbohydrates and fats. Furthermore, it can control the level of blood glucose. Thus, the concentration of insulin in blood has been used as an indicator for diagnosis of diseases such as diabetes. Therefore, developing a rapid and reliable analytical method for determining insulin level in sera is essential. We recently have generated dextran encapsulated gold nanoclusters (AuNCs@Dextran) from one pot reactions. In this work, we discovered that the AuNCs@Dextran have selective binding affinity toward insulin. For ease of isolation, the AuNCs@Dextran were immobilized on the surface of the Fe3O4 MNPs and used as affinity probes for insulin. Insulin is composed of A and B chains, linked by disulfide bonds. After using the reduced insulin containing free forms of A and B chains as the sample, it was discovered that the insulin B chain can be selectively trapped by the AuNCs@Dextran-Fe3O4 MNPs. Insulin was further digested using trypsin to generate peptide residues. The resultant tryptic digest was incubated with the AuNCs@Dextran-Fe3O4 MNPs for conducting selective trapping experiment followed by characterization by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS). The results showed that only the peptide (#1-22) from B chain appeared in the resultant mass spectrum, indicating that the peptide is the binding site to the AuNCs@Dextran. Additionally, three synthetic peptides derived from this peptide sequence (#1-22) were further used as the model sample for selective enrichment by the AuNCs@Dextran-Fe3O4 MNPs. The peptides trapped by the AuNCs@Dextran were characterized by the MALDI-MS. The results showed that only the ion peak with the sequence (#8-18) derived from B chain appeared in the MALDI mass spectrum, indicating this peptide residue is the binding site to the AuNCs@Dextran. The results showed that limit of detection of this approach against insulin using MALDI-MS as the detection method was as low as ~ 1 pM. Since insulin level in blood is usually in several tens to several hundred pM, this approach is potentially suitable to be used in determination of insulin in real samples such as sera. In the end of this study, quantitative analysis using this approach for determination of insulin in sera was also studied. The error was ~ 16%, indicating the potential of using this approach in real world applications.