利用功能性奈米粒子及凝血酶偵測血小板生長因子及免疫球蛋白G

Abstract

蛋白質檢測極為重要，並可應用於臨床診斷、癌症、病原體感染和遺傳性疾病的研究。我們利用比色法開發出兩種具有高選擇性和高靈敏度的蛋白質生物感測器，並使用磁性奈米粒子與兩種金奈米粒子—核酸適體修飾的13奈米金粒子(Apt–Au NPs)，和纖維蛋白原吸附的金奈米粒子(Fib–Au NPs, 56 nm)等來檢測血小板生長因子(platelet-derived growth factors, PDGF)以及人類免疫球蛋白G (hIgG)。在檢測PDGF的系統中，Apt–Au NPs有辨識目標分子的功能，而Fib–Au NPs則是扮演reporting units。在此系統中，功能性金奈米粒子(AptPDGF/Aptthr29–Au NPs)上面有與血小板生長因子結合的核酸適體(PDGF-binding-aptamer, AptPDGF)，以及與凝血酶結合的核酸適體(thrombin-binding-aptamer, Aptthr29)。凝血酶會催化修飾在金奈米上的血纖維蛋白原，形成不溶的纖維素，因此造成了金奈米粒子的聚集，若凝血酶的活性被AptPDGF/Aptthr29–Au NPs抑制，則不會造成聚集。如果溶液中有PDGF先與AptPDGF/Aptthr29–Au NPs結合，則凝血酶就因立體障礙而無法接至AptPDGF/Aptthr29–Au NPs表面。此系統偵測PDGF的線性範圍是0.5–20 nM (R2 = 0.96)，在100 μM的牛血清蛋白存在下，偵測極限是0.3 nM。若把 AptPDGF/Aptthr29–Au NPs用來濃縮樣品中的PDGF，那麼偵測極限可降至35 pM。最後我們測定乳癌細胞培養基中的PDGF濃度為230 (±20) pM，證明此方法是一個簡單，具有高專一性和靈敏度的方法。第二個主題是將類似的概念應用於hIgG的檢測。第一步先將hIgG與修飾protein G (PG)的磁性奈米粒子(PG–MNPs)還有PG修飾的Apt–Au NPs (PGApt–Au NPs)結合，並用磁鐵分離它們的複合體(PG–MNPs…hIgG…PGApt–Au NPs)。第二步是利用上清液中沒有被磁鐵分離的PGApt–Au NPs來控制凝血酶的活性，因此越多hIgG，會形成越多的PG–MNPs…hIgG…PGApt–Au NPs複合體，上清液中和凝血酶結合的PGApt–Au NPs也就越少，使fibrin–Au NPs的聚集越嚴重。在最佳化的條件並且含有100 μM的牛血清蛋白之下，PG–MNPs/PGApt–Au NPs/Fib–Au NPs probe可將偵測極限降至5 nM 。 我們最後取得三個正常人的血液樣本與兩個類風濕關節炎病人的血液樣本，利用標準曲線測得其hIgG濃度，並和市售的儀器做比較(enzyme-linked immunosorbent assay)，發現兩者的數值呈現良好的線性(R2 = 0.98)，證明了此法的實用性與準確性。

Protein detection is of great importance in basic research and clinical diagnosis of genetic disorders and its associated diseases, cancers, and pathogen infections. We have developed two colorimetric protein sensors using aptamer modified 13-nm gold nanoparticles (Apt–Au NPs), thrombin, and fibrinogen adsorbed Au NPs (Fib–Au NPs; 56 nm). These could be used for the highly selective and sensitive detection of platelet-derived growth factors (PDGFs) and human immunoglobulin G (hIgG). In the PDGF system, Apt–Au NPs and Fib–Au NPs were the recognition and reporting units, respectively. PDGF-binding-aptamer (AptPDGF) and 29-base-long thrombin-binding-aptamer (Aptthr29) were conjugated with Au NPs to prepare functional Apt–Au NPs (AptPDGF/Aptthr29–Au NPs) for specific interaction with PDGF and thrombin, respectively. Thrombin interacted with Fib–Au NPs in solution to catalyze the formation of insoluble fibrillar fibrin–Au NPs agglutinates through the polymerization of unconjugated and conjugated fibrinogen. Thrombin activity was suppressed when it interacted with AptPDGF/Aptthr29–Au NPs due to steric effects through the specific interaction of PDGF with AptPDGF on the surfaces of AptPDGF/Aptthr29–Au NPs. Under optimal conditions with AptPDGF/Aptthr29–Au NPs at 25 pM, thrombin at 400 pM, and Fib–Au NPs at 30 pM, AptPDGF/Aptthr29–Au NPs/Fib–Au NPs probe responded linearly to PDGF over a concentration range of 0.5–20 nM with a correlation coefficient of 0.96. The limit of detection (LOD, signal-to-noise ratio = 3) for each of the three PDGF isoforms was 0.3 nM in the presence of bovine serum albumin at 100 μM. When using AptPDGF/Aptthr29–Au NPs to selectively enrich PDGF and remove interfering substances from cell media, LOD of this probe for PDGF was 35 pM. This probe revealed that the concentration of PDGF in the three cell media is 230 (±20) pM, showing its advantages in terms of simplicity, sensitivity, and specificity. We also developed a method for the selective IV and sensitive detection of human immunoglobulin G (hIgG). The first step involves the specific interactions of hIgG with protein G (PG)-functional Fe2O3 magnetic NPs (PG–MNPs) and with PG– and aptamer (Apt)– modified gold NPs (PGApt–Au NPs) and the subsequent magnetic separation of their complexes (PG–MNPs…hIgG…PGApt–Au NPs). In the second step, the concentration of free PGApt–Au NPs was determined by taking advantage of their control of thrombin activity toward fibrinogen-modified Au NPs (Fib–Au NPs). The activity of thrombin toward Fib–Au NPs to form fibrin–Au NP aggregates was inhibited by PGApt–Au NPs through the specific interaction of thrombin with the Apt. The greater the amount of hIgG in a sample, the less free PGApt–Au NPs remained in the supernatant. Consequently, greater amounts of free thrombin remained, which led to the formation of greater amounts of fibrin–Au NP aggregates. Under optimal conditions (8 μg/mL PG–MNPs, 1.0 nM PGApt–Au NPs, 400 pM thrombin, 30 pM Fib–Au NPs), PG–MNPs/PGApt–Au NPs/Fib–Au NPs probe allows the selective detection of hIgG down to 5 nM in the presence of 100 μM of BSA. The practicality of this approach was validated by determining the concentrations of hIgG in spiked plasma samples that were in good agreement with determinations made by enzyme-linked immunosorbent assays (R2 = 0.98). These results demonstrate that this assay has great potential for diagnosing diseases associated with changes in hIgG levels.