Article pubs.acs.org/ac
Assembly of Selective Biomimetic Surface on an Electrode Surface: A Design of Nano−Bio Interface for Biosensing Tao Gao,† Fengzhen Liu,‡ Dawei Yang,† Yue Yu,§ Zhaoxia Wang,‡ and Genxi Li*,†,∥ †
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, People’s Republic of China ‡ Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, People’s Republic of China § Department of Hepatobiliary Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University, Nanjing 210008, People’s Republic of China ∥ Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, People’s Republic of China S Supporting Information *
ABSTRACT: In nature, cellular molecule sensing is usually achieved at the environment/membrane interface. In the meantime, rapid growth of nanotechnology is increasingly pushing engineered nanomaterials to interact with biological surfaces. Herein, inspired by trans-membrane signal transduction, a nano−bio interface has been constructed in this work for biosensing application. The interface is formed between a selective biomembrane mimetic surface (SBMMS) and a function-oriented 2D nanohybrid. Based on the design, target recognition can be performed in a biologically favorable environment, and the nano−bio interaction can be transduced into amplified electrochemical readouts. Furthermore, this sensing platform can be used to analyze various kinds of targets, including proteins, nucleic acids, and small molecules, just by changing the biorecognition element. Low detection limits and wide detection ranges can also be obtained. So, this nano−bio interface may provide a new platform for bioanalytical research in the future.
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discussed.23 Based on these works, we are considering fabricating a biologically favorable surface for biosensor design. Furthermore, greatly inspired by cell signal transduction in biological systems, which convert a molecular recognition event at the cell surface to in vivo cascade biochemical responses,24 we may transform the biorecognition event at a modified electrode surface into electrochemical readouts. The interface of nanomaterial with biology represents one of the fastest growing and most promising areas of nanotechnology. The interface includes those fabricated with proteins,25−28 DNA,29−32 carbohydrates,9,33,34 organelle,35,36 and cells.37,38 Understanding of bio-physicochemical interaction at the nano−bio interface39,40 has provided unprecedented opportunity for biosensor development. However, there are two major issues to be solved. One is how to reduce the influence of nanomaterial on biorecognition when playing different roles.25,41−43 The other is how to engineer nanomaterial to control its function to interact with a biosurface.40,44 Keeping the two major issues in mind, we are endeavoring to manipulate function-oriented nanomaterials with synergistic
iosensors are analytical devices that may transform the biorecognition signal into another measurable signal. A biosensor usually includes three parts: biorecognition element, transducer, and read device.1,2 Since it is a great difficulty to to sensitively and selectively capture a specific bioanalyte in complex biological environments, such as peripheral liquids, cell lysates, or even tissues, careful choice and design of the biorecognition element is vitally important. In the meantime, as the transducers used in biological assays are usually based on biological binding pairs of antibody−antigen,3,4 receptor− ligand,5,6 enzyme−substrate, 7,8 and multivalent recognition,9−11environments for the bindings that may facilitate their interactions are especially essential.12,13 So, how to assemble a biorecognition element with a biologically favorable environment is most desirable for sensor fabrications, yet it is still a challenge. Surface modification is playing increasingly important roles in biosensor design,14 and recent developments in biomolecular modification have further enabled us to interrogate biointeractions.15−17,39,42 As described in our previous work, we have respectively studied the interplay of β-amyloid peptides,18 ionchannel protein,19 or protein enzymes20−22 with biomimetic surface (self-assembled lipid bilayer on electrode). Meanwhile, the interplay between gold nanoparticles and selective surface (self-assembled DNA monolayer on electrode) has also been © XXXX American Chemical Society
Received: March 2, 2015 Accepted: April 30, 2015
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DOI: 10.1021/acs.analchem.5b00816 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
resolution transmission electron microscopy (HRTEM) observations were carried out on Hitachi H7650 and JEOL 2200FS instruments, with accelerating voltages of 80 kV and 200 kV, respectively. The specimens were prepared by dispersing the GO−AgNPs hybrid on 400 mesh size copper grids. Electrode Treatment and Modification. Gold disk electrode (3 mm in diameter) was cleaned with piranha solution (H2SO4:H2O2 = 3:1, v/v) for 5 min, followed by rinsing the electrode with sufficient double-distilled water. The electrode was then polished on a microcloth with alumina powder of 1.0, 0.3, and 0.05 μm in sequence. Subsequently, the electrode was treated by ultrasonication separately in both ethanol and double-distilled water for 5 min. Then, the electrode was immersed in 50% HNO3 for 30 min, followed by electrochemically cleaning with 0.5 M H2SO4 to remove any remaining impurities. After being dried with high purity N2, the preceding gold electrode was immediately used for E-TBA immobilization. The electrode was incubated with 0.1 μM ETBA (hybridized with its 5′ complementary strand) in DNA modification solution for 10 h at room temperature. The gold electrode was rinsed with sufficient buffer to remove unbound E-TBA before the following experiments. The assembly of lipid bilayer on gold electrode was based on our previous work.46 First, the pretreated gold electrode was immersed into an ethanol solution containing 2 mM DPPTE overnight at room temperature to form the first layer of the lipid bilayer, followed by rinsing the electrode with ethanol to remove the noncovalent binding DPPTE. DPPTE modified electrode was then immersed in a solution (decane:ethanol = 3:1) containing 25 mM DPPC for 5 min. Then, the electrode was cooled to −20 °C to form the second layer of the lipid bilayer, followed by immersing the electrode in 0.1 M KCl for 2 h. The lipid bilayer membrane anchored with a small amount of DNA aptamers was then formed on gold electrode. The GO modified glassy carbon electrode (GO/GC) and GO−AgNPs modified GC electrode (GO−AgNPs/GC) were prepared as follows: 5 μL of GO suspension (5 μg mL−1) or GO−AgNPs suspension (5 μg mL−1) was cast on a polished glassy carbon electrode. The electrode was then dried under an infrared lamp, followed by dropping 2 μL of 0.1% Nafion solution as a binder. The electrode was dried again under the infrared lamp. Finally, it was rinsed with deionized water for several times before use. Electrochemical Measurements. All electrochemical measurements were performed on a CHI660D Potentiostat (CH Instruments) workstation. The EIS and DPV were obtained by using a three-electrode system, which involved the premodified gold disk electrode as the working electrode, a saturated calomel electrode (SCE, saturated with KCl) as the reference electrode, and a platinum pillar as the counter electrode, respectively. The EIS experimental parameters were as follows: bias potential, 0.225 V; amplitude, 5 mV; frequency range, 0.01 Hz to 100 kHz. DPVs were recorded under the experimental parameters: initial potential, −0.05 V; final potential, 0.20 V; amplitude, 5 mV; pulse period, 0.2 s. All electrolytes were bubbled with high purity N2 to avoid the influence of dissolved O2. Human α-thrombin, a serine protease that is crucial in pathological coagulation disease,47 was selected as the model target in this work. For the detection of thrombin protein, the SBMMS modified gold electrodes were cast with 5 μL binding buffer containing different concentrations of human α-
effects for biosensor design. So, in this work, a selective biomembrane mimetic surface (SBMMS) is first fabricated at an electrode surface for bioanalyte recognition. The SBMMS is composed of lipid bilayer and DNA aptamer, which can provide unique binding environments for specific bioanalytes. Then, a function-oriented 2D nanohybrid, composed of graphene oxide and silver nanoparticles (GO−AgNPs), is synthesized as the transducer to interplay with the SBMMS. Moreover, the nanohybrid can also be used as an ideal signal amplification element through the solid-state electrochemical stripping technique. Further to our satisfaction, electron transfer at the interface is enhanced by dual interactions between GO−AgNPs and the SBMMS. With the superior properties of this fabricated nano−bio interface between GO−AgNPs and the SBMMS, a sensitive and label-free sensing platform has been constructed for bioanalytical assays.
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MATERIALS AND METHODS Reagents and Chemicals. Chemically exfoliated GO was obtained from Nanjing XFNANO Materials Tech Co., Ltd. and was further purified by ultrasonic wash and centrifugation filtering. The human α-thrombin (Thr), human serum albumin (HSA), myoglobin, silver nitrate (AgNO3), sodium borohydride (NaBH4), hydroquinone (HQ), and 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) were obtained from SigmaAldrich. 1,2-Dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) was purchased from Avanti Polar Lipids, Inc. The vascular endothelial growth factor 165 (VEGF165) was from ZhongKeWuYuan Biotechnology Co., Ltd. The exonuclease I was obtained from Takara Co., Ltd. (Dalian, China). The fetal calf serum (FCS) was purchased from Biological Industries (Beth Haemek, Israel). Other chemicals were all of analytical grade and were used without further purification. All of the DNA sequences used in this study were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences were as follows: 5′ thiol group functionalized thrombin binding aptamer (E-TBA), 5′-SH-C12TTTTTTTTTTTTTTTGGTTGGTGTGGTTGG-3′; its 5′ complementary strand, 5′-AAAAAAAAAAAAAAA-3′; thrombin binding aptamer (TBA), 5′-GGTTGGTGTGGTTGG-3′. The buffer solutions used in this work were as follows: TE buffer for DNA storage, 10 mM Tris-HCl and 0.1 mM EDTA, pH 7.4; solution for DNA modification, 10 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, and 1 μM TCEP, pH 7.4; DNA binding solution, 10 mM phosphate buffer and 10 mM NaCl, pH 7.2; thrombin binding buffer, 10 mM Tris-HCl, 100 mM NaCl, and 5 mM KCl, pH 7.4 (it was also used as the electrolyte for differential pulse voltammogram (DPV)). The buffer for electrochemical impedance spectra (EIS) was 5 mM [Fe(CN)6]3−/ 4− and 0.5 M KNO3. All solutions were prepared with deionized water, which was purified with a Milli-Q purification system (Bedford, MA, USA) to a specific resistance of 18.2 MΩ. Synthesis and Characterization of GO−AgNPs. GO− AgNPs were synthesized according to the previous report.45 Samples for characterization were dispersed in deionized water. Ultraviolet−visible absorption spectra (UV−vis) were recorded on a Shimadzu UV-2450 spectrophotometer. Raman spectra were obtained by using a JY LabRam HR800 UV micro-Raman system with the 514.5 nm line of an Ar+ laser as the excitation source. The parameters were as follows: focus area,
