基于金納米粒子的新型電化學(xué)適體生物傳感器信號放大檢測癌胚抗原

1、Electrochemistry Communications 37(20131519Contents lists available at ScienceDirectElectrochemistry Communicationsj o u r n a l h o m e p a g e :w w w. e l s e v i e r. c o m/l o c a t e /e l e c o mShort communicationNovel electrochemical aptamer biosensor based on gold nanoparticles signal ampli

2、cation for the detection of carcinoembryonic antigenHuawei Shu, Wei Wen, Huayu Xiong, Xiuhua Zhang, Shengfu Wang Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules &

3、College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, PR Chinaa r t i c l e i n f o a b s t r a c tA novel electrochemical aptamer biosensor was designed based on the signal ampli cation of gold nanoparticles (AuNPsfor the detection of a tumor biomarker, carcinoembryonic ant

4、igen (CEA.The electrochemical biosensor was constructed by sandwiching the CEA between an Au electrode modi ed with thiol-terminated CEA aptamer-1(Apt1and the AuNPs with thiol-terminated CEA aptamer-2(Apt2and 6-ferrocenyl hexanethiol (Fc.Amper-ometric detection of Fc by differential pulse voltammetr

5、y (DPVon the electrochemical biosensor was used to quantify the concentration of CEA. The biosensor provided a linear range from 1to 200ng/mLfor CEA with a de-tection limit of 0.5ng/mL.Its performance was successfully evaluated with human serum spiked with CEA, indi-cating that the aptasensor has gr

6、eat potential for practical application. In addition, the electrochemical biosensor exhibited excellent selectivity responses and good stability toward the target analyte.©2013Elsevier B.V. All rights reserved.Article history:Received 15August 2013Received in revised form 7September 2013Accepte

7、d 17September 2013Available online 5October 2013Keywords:Carcinoembryonic antigen AptamerElectrochemical biosensor Gold nanoparticles1. IntroductionCarcinoembryonic antigen (CEAis a 180kDa highly glycosylated protein over-expressed on breast, colon and other cancer cells 1. Argu-ably, it is the best

8、 studied tumor epitope and present on the largest num-ber of tumors. These, and other conditions, lead to an increase in blood CEA. Hence, clinically, serum CEA levels may be indicative (butnot diag-nostic of the return of active metastatic disease 2. The usual technique used for the determination o

9、f tumor markers is immunoassay. Immuno-assays, including radio immunoassays 3, enzyme immunoassays 4, uoro immunoassays 5, and piezoelectric immunosensors 6, have al-ready been reported for CEA. But aptamers (Aptshave many advantages over antibodies in terms of repeatable synthesis, easy modi cation

10、, long-term stability, less immunogenicity etc., and are increasingly used in bioanalysis and biotechnology 7,8.Apts are single-stranded DNA or RNA molecules. They have been se-lected by SELEX (systematicevolution of ligands by exponential enrich-ment technology from a combinatorial library by their

11、 capability to bind a speci c target such as a nucleic acid, protein or tumor cell 911. There are two main approaches to constructing electrochemical aptasensors. The rst makes use of the fact that aptamers can readily undergo site-speci c modi cation during chemical or enzymatic syn-thesis to incor

12、porate particular reporters, linkers, or other moieties. Al-ternatively, aptamer secondary structures can be engineered to undergo analyte-dependent conformational changes. This, combined with theCorresponding author. Tel.:+862750865309; fax:+862788663043. E-mail address:wangsf(S. Wang. 1388-2481/$s

13、ee front matter ©2013Elsevier B.V. All rights reserved. /10.1016/j.elecom.2013.09.018ability to speci cally place chemical agents, opens up a wealth of possi-ble signal transduction schemas 12. Electrochemical approaches using aptamers in analytical applications have become incr

14、easingly popular over the past two decades 13,14because electrochemical techniques have high sensitivities, easy operation and low cost, and are suitable for miniaturization.So far, CEA Apts have been reported 15. However, CEA usually binds to one copy of Apt. In this work, we report a novel voltamm

15、etric assay using Fc capped AuNPs Apt2conjugates for the analysis of CEA and Apt1in the form of sandwich complexes. Because two different Apts are used simultaneously for CEA identi cation, the identi cation accuracy is higher than with a single aptamer, resulting in a low back-ground signal and goo

16、d selectivity. 2. Experimental sections 2.1. Reagents and apparatusHydrogen tetrachloroaurate (IIItrihydrate (HAuCl4·3H2O, tri-sodium citrate, 6-ferrocenyl hexanethiol (Fc,CEA from human uids (95%,SDS-PAGE, human serum and 6-mercapto-1-hexanol (MCH,tris(2-carboxyethyphosphine hydrochloride (TCE

17、Pwere purchased from Sigma-Aldrich (USA.All other reagents were of analytical grade. Oligonucleotides were synthesized and puri ed by Sangon (Shanghai,China. The sequences of these oligonucleotides used in this work are listed as follows:Apt1:3-SH-ATACCAGCTTATTCAATT-5; Apt2:3-SH-AGGGGGTGAAGGGATACCC-

18、5.16H. Shu et al. /Electrochemistry Communications 37(20131519The electrochemical measurements were carried out on a CHI 660C electrochemical working station (CHInstruments, Inc. Shanghai using a three-electrode system. 2.2. Preparation of AuNPsAuNPs were prepared according to the method reported pr

19、eviously 16. 2.7mL of 1%trisodium citrate was added to 100mL of boiling 0.01%HAuCl 4solution and stirred for 10min at the boiling point. The prepared colloid AuNPs were stored in brown glass bottles at 4°C.The nal AuNPs had an average diameter of approximately 13nm as measured by a Transmission

20、 Electron Microscope (TEMas shown in Fig. 1A. 2.3. Preparation of Fc capped AuNPs Apt2conjugatesThe modi cation of AuNPs with Fc and Apt2was prepared according to the method reported by J. Wang et al. 17. A 500L colloid gold solu-tion was mixed with 100L of hexane containing 5.0mM Fc and 200L Apt2fo

21、r 24h on a vortex stirrer. The resultant modi ed AuNPs remained in the aqueous phase. The solution was centrifuged, and the hexane phase containing Fc was decanted. The AuNPs capped with Fc and Apt2were thoroughly rinsed with 500L hexane, resuspended in 500L of 0.1mol/Lphosphate buffered saline (PBS

22、,pH 7.0. Following centrifugation of the mixture to remove the supernatant, the resulting Fc capped AuNPs were washed and redispersed in 500L PBS and stored at 4°Cfor further use.2.4. Fabrication of the biosensor and the electrochemical detection The bare Au electrode was polished with 0.05mm a

23、lumina slurries and ultrasonically cleaned in ethanol and ultrapure water twice for 5min. The Au electrodes were then put in 5%H 2SO 4solution and scanned from 0to 1.6V to measure the cyclic voltammetry (CVsignals until a standard cyclic voltammetric peak appeared. After washing with ultrapure water

24、 and drying with a stream of N 2, the pre-cleaned Au electrode was incubated for 1h at room temperature in 50L of 1M Apt1including 10L of 5M TCEP and washed with ultrapure water. Then the electrode was immersed in 50L of 1mM MCH for 1h to obtain a well-aligned DNA monolayer. Next, this electrode was

25、 immersed in 50L CEA solution (solutionin 0.1M PBS, pH 7.4 for 30min, rinsed with PBS to remove the nonspeci cally bound CEA, and was then imme-diately soaked in 50L Fc capped AuNPs Apt2conjugates for another 30min. The electrode above was in contact with PBS and scanned from 0to 0.6V to measure the

26、 DPV signals. The whole preparation pro-cess is outlined in Fig. 2. 3. Results and discussion3.1. Characterization of biosensor fabricationElectrochemical impedance spectroscopy (EISand CV measure-ments were used to characterize the electrochemical biosensor 18. In the terms of EIS, Fe(CN63/Fe(CN64w

27、as utilized as the redox probe and the semicircle diameter was equal to electron-transferA1800016000 140001200010000800060004000200010000200003000040000500006 420-2-4-6-20.100.080.060.040.020.00D0.04:12:14:31:12:3Potential / V/4Fig. 1. Characteristics of AuNPs TEM (Aand

28、 EIS(Band CV(Cin 0.5mM Fe(CN3at bare Au (a,Apt1/Au(b,CEA/Apt1/Au(cand Fc AuNPs Apt2/CEA/Apt1/Au(delectrodes. Op-6timization of experimental condition for DPV response to the different volume proportions of AuNPs modi ed Fc (5mM and Apt2(1M (D.H. Shu et al. /Electrochemistry Communications 37(2013151

29、917HSMCHAu0.400.350.30aC u r r e n t /u A50.100.05bb0.000.0Potential/VSHAU NPsAU NPsSHCEA MCHSHSH-CEA aptamer-1SH-CEA aptamer-26-ferrocenylhexanethiolFig. 2. Schematic diagram about the electrochemical aptamer biosensor based on AuNPs signal ampli cation for the detectio

30、n of CEA./4resistance. In 0.5mM Fe(CN3, bare electrode exhibited almost a 6straight line (Fig. 1B, curve a, which was characteristic of a mass diffu-sion limiting step of the electron-transfer process. When the Apt1was self-assembled on to the bare electrode via Au S binding, the R et in-creased (Fi

31、g. 1B, curve b, this was because that the negatively charged phosphate backbone of the oligonucleotides produced an electrostatic repulsion force to Fe(CN63/Fe(CN64. It is also found that the as-sembly of CEA on the Apt1modi ed electrode leads to a signi cant increase in R et (Fig. 1B, curve c. Afte

32、r hybridization with Apt2AuNPs Fc, there is a large increase in R et (Fig. 1B, curve d, implying that the introduction of Apt2AuNPs Fc greatly inhibits the electron transfer of the redox probe on the electrode surface 19. These results were in a good agreement with those obtained from CV measurement

33、s (Fig. 1C. As the above gure shows, both results of EIS and CV demon-strate that the sensing interface has been fabricated successfully.3.3. Optimization of incubation timeIt was found that changing the incubation time of the Apt1modi ed electrode (Fig. 3B, curve a and Apt2AuNPs Fc (Fig. 3B, curve

34、b in CEA solution (50ng/mLcaused a visible difference in the increase of peak current. Therefore, the dependence of CEA incubation time on the in-crease of the peak current was studied to determine the optimum incu-bation time of CEA. As shown in Fig. 3B, the current response increased with the incr

35、ease of incubation time and almost leveled off after 30min, when the building of the Apt1CEA Apt2AuNPs Fc complex reached saturation. Therefore, 30min was chosen as the incubation time for the detection of CEA. 3.4. Performance of aptasensor3.4.1. Calibration curve of aptasensorThe aptasensors were

36、incubated in different concentrations of CEA under the optimal conditions and the DPV responses of the proposed aptasensor were recorded. As seen in Fig. 3C, which displays the corre-sponding calibration plots, the current was proportional to CEA concen-tration over the range from 1to 200ng/mL.The l

37、imit of detection was 0.5ng/mL(S/N=3. The linear equation could be tted as I (A=1.68E 8+0.9373c (g/mL(R=0.9978, which is lower than the de-tection limit of 1.1ng/mL20obtained using immunosensors. 3.4.2. Speci cityThe speci city of the proposed aptasensor was also examined by detecting the DPV change

38、 in the presence of CEA and three interfering agents:myoglobin (MYO,mucoprotein (MUC,and bovine serum al-bumin (BSA.The experimental results are shown in Fig. 3D:CEA (100ng/mLproduced a much stronger current response, while the other interfering agents (1g/mLcaused almost negligible electro-chemical

39、 changes.3.2. Optimization of experimental conditionsAs shown in Fig. 1D, when the ratio was less than the best propor-tion, because the amount of Fc was less, the DPV signal was relatively low; when the ratio was greater than the best, because the quantity of Apt2was less, affecting the amount of A

40、uNPs compounds combined with the CEA, there is a decrease in the electrochemical signal. There-fore, the optimized volume proportion of AuNPs modi ed Fc (5mM and Apt2(1M was determined to be 1:2.In the optimized volume proportion of AuNPs modi ed Fc (5mM and Apt2(1M, the dosage of the Apt2determined

41、 the ef ciency with which the Apt2binds to CEA, and would affect the electrochemical signal. As shown in Fig. 3A, with an increase in the amount of Apt2, the signal intensity gradually increased and then was nearly balanced when the amount of Apt2was 200L. Therefore, the optimal dosage of the Apt2wa

42、s 200L.18H. Shu et al. /Electrochemistry Communications 37(201315190.070.060.050.040.03Bab0.020.010.000102030405060V CEA-apt 2/µL Time/min0.16D0.120.080.040.00CEA MYO MUC BSAFig. 3. Optimization of experimental condition for DPV response to 1M Apt2of different volumes (Aand DPV peak currents pl

43、otted against incubation time of the Apt1modi ed elec-trode (aand Apt2AuNPs Fc compounds (bin CEA (50ng/mLsolution at 37°C(B.DPV of 1, 5, 10, 25, 50, 75, 100, 150, and 200ng CEA in the 0.1M PBS (pH7.0. Insert:linear rela-tionship between the increase peak current and the concentration of CEA. T

44、he selectivity of the aptasensor:CEA (100ng/mL,MYO (1g/mL,MUC (1g/mL,BSA (1g/mL(D.3.5. Analytical application of the proposed aptasensorTo evaluate the applicability of the proposed aptasensor, the concen-tration of CEA in human blood serum sample was determined by the standard addition method. In t

45、his test, 30L of original human serum was diluted to 3.0mL with PBS. Then different amounts of CEA (100,120, 140ng were added in 1mL diluted human serum samples to pre-pare three serum specimens containing CEA. Each sample was detected three times. The detection value was the average of three result

46、s. The analytical results are shown in Table 1. The recovery was in the range 95.6101.0%,indicating that the aptasensor had good accuracy and had great potential for the analysis of CEA in real clinical samples. 4. ConclusionIn summary, a novel electrochemical aptasensor for the detection of CEA has

47、 been developed. Unlike most existing electrochemical aptasensors, two different Apts were used simultaneously for CEA iden-ti cation by using Apt2AuNPs Fc conjugates for the analysis of CEAand Apt1in the form of sandwich complexes. This produced better iden-ti cation accuracy than when using a sing

48、le aptamer, resulting in a low background signal and good selectivity. This electrochemical aptasensor was easy to produce, and might prove promising for applications in bi-ological or clinical target analysis. AcknowledgmentsThis work was supported by the National Natural Science Founda-tion of China (No.21175032 and the Natural Science Fund for Creative Research Groups of Hubei Province of China (No.2011CDA111. References1V. Kulasin