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Electrophoresis-enhanced localized surface plasmon resonance sensing based on nanocup array for thrombin detection
Sensors and Actuators B 232 (2016) 219–225
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrophoresis-enhanced localized surface plasmon resonance sensing based on nanocup array for thrombin detection Shuang Li a,b , Diming Zhang a , Qian Zhang a , Yanli Lu a , Nantao Li a , Qunwei Chen c , Qingjun Liu a,b,∗ a Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, 310027, PR China b Collaborative Innovation Center of Health Management, Fujian University of Traditional Chinese Medicine, Fuzhou, 350108, PR China c Zhejiang Chinese Medical University, 310053, PR China
a r t i c l e
i n f o
Article history: Received 25 December 2015 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online 25 March 2016 Keywords: Localized surface plasmon resonance (LSPR) Electrophoresis Nanocup array Thrombin Biosensor
a b s t r a c t Based on the special electrical and optical properties resulted from periodic nanostructure, localized surface plasmon resonance (LSPR) sensors have been widely used for various chemical and biological detections. Utilizing uniform alignment nanocups, an electrophoresis-enhanced LSPR sensor was designed for sensitive thrombin detection. The nanocup array was composed of nano-scaled funnel shaped cups with nanoparticles along the side walls. Through self-assembly, polyethylene glycol, thrombin-specific peptide, and bovine serum albumin were successively immobilized on the nanocup array. The results demonstrated that the synchronous implementation of electrophoresis and LSPR measurement could lead to obvious peak shifts in optical transmission spectra for detecting thrombin. The detection limit was as low as 10−11 M. With the high sensitivity and well linearity, the electrophoresisenhanced LSPR sensing offered a novel design perspective for chemical and biological sensors with specific modifications on the nanosensor surfaces. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Recent progresses in highly sensitive optical transducers, which combined with the exquisite specificity, affinity, and versatility of bio-molecular interactions, have greatly promoted the development of a wide variety of optical biosensors in the field of biomedical and environmental science. Among these sensors, localized surface plasmon resonance (LSPR) was one of the most widely utilized techniques in optical biosensors design to measure molecular binding kinetics by the virtue of density oscillations of conduction electrons on the metallic nanostructure surfaces, which varied with the affinity of the analytes [1–3]. The mechanism of these LSPR sensors was often based on the changes of transmission spectra, which responding to refractive index changes from the binding of molecules on the nanoparticle surfaces. Typically, the increase of refractive index would lead to the red shift. While, the decrease of refractive index would lead to the blue shift in spectral absorption and scattering peaks [4–8].
∗ Corresponding author. E-mail address: [email protected] (Q. Liu). http://dx.doi.org/10.1016/j.snb.2016.03.134 0925-4005/© 2016 Elsevier B.V. All rights reserved.
LSPR phenomenon on nanostructured devices (e.g. nanoparticles, nanoholes, and nanorods) had high sensitivities in detecting small changes in refractive index of analytes. However, the difficulties in the size control of the nanoparticles and the multiple scattering peaks of the nanoholes were often the obstacles on the LSPR detection. Lycurgus cup can appear different colors under the condition of different light illumination directions. According to some publications, it was due to metal nanoparticle optical scattering and inspired the research in the field of nanoplasmonics [9–11]. Giving rise to the effect of the Lycurgus cup structure, nanocup array is characterized by transmission peaks in the visible wavelength and has a high sensitivity in detecting small changes in refractive index of analyte. Some groups have reported that the hybrid structures of nanocup array with nanoparticles have a more stable optical performance in transmission spectra, which could be used to improve stabilities of the LSPR detections [12,13]. On the other hand, diffusion of the target molecules was also often limited during the mass transportation process on the plasmon resonance surface, which could slow down the molecular binding velocity and thereof limit the sensitivity of sensors. In order to improve the efficiency of mass transportation, electrokinetic preconcentration of charged molecules toward sensing surface could accelerate the analytes binding, which have been studied by several
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groups [14–16]. At the same time, the intensity of LSPR could also be influenced by electron densities and electrical attractions in the electrochemical scanning, which could enhance the LSPR signals [17]. The electrochemical scanning could change redox reaction conditions around the nanosensor surface, elicit refractive index changes, finally generate extra LSPR wavelength shifts [13]. Therefore, the combination of electrophoresis and LSPR may provide a promising approach to enhance LSPR signals with significant resonance wavelength shifts in transmission spectra for analyte detections. Thrombin is a serine protease that plays an important role in blood coagulation, thrombosis, inflammation, angiogenesis, tumor growth and metastasis [18,19]. Various sensing methods have been reported for the detection of thrombin, such as quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and electrochemical analyses, which were mostly on the basis of thrombin aptamers [20,21]. In order to improve the specificity of the detection, self-assembled peptides have been considering as novel approaches for modification of sensors in biological detection [22,23]. Without the redundant structures, peptides could result in the capability for practical applications through simplifying the manufacture. Thus, the thrombin specific cleavage peptides have been used to fabricate ultrasensitive thrombin biosensors to study the mechanistic details of cleavage processes [24]. In this paper, an electrophoresis-enhanced LSPR measuring method based on peptide modified nanocup array was used as the nanosensor for thrombin detection. The surface of the nanocups was modified with a sandwich structure including the polyethylene glycol (PEG), thrombin specific peptide, and bovine serum albumin (BSA). By applying direct current onto the electrodes, the transport of molecules toward the sensor surfaces was accelerated and the resonance wavelength shifts in the transmission spectra were enhanced significantly. The electrophoresis-enhanced LSPR sensing method might provide a promising approach for sensitive and selective thrombin detection.
2. Experiments 2.1. Electrophoresis-enhanced LSPR for thrombin detection The schematic of the experimental setup is shown in Fig. 1A. The halogen cold laser was delivered through the nanosensor and indium tin oxide (ITO) electrode, finally received by a CCD camera. The direct current was applied between the nanosensor and ITO electrodes, which were utilized as the positive and negative electrodes, respectively, for electrophoresis enhanced LSPR measurement. In the measurement, the gap distance between the ITO electrode and the nanosensor was fixed at 1 cm. As shown in Fig. 1B, a “sandwich” strategy was employed. Polyethylene glycol (PEG) used to form a membrane on the nanosurface for better immobilization of the peptide. The thrombin specific peptide was used to determine the thrombin catalytic activity. The negative charged bovine serum albumin (BSA) was added at the terminus of the peptides to amplify the LSPR signals by electrophoresis, for which could accelerate the peptide’s cleavage reaction. The thrombin solution was introduced into the nanosensor surface, which was followed by thrombin cleavage at its specific site. With direct current applied between the ITO electrode and the nanosensor surface, the cleaved peptide and electronegative BSA would be separated. In the meantime, due to the refractive index changes on the sensor surface, significant violet-shift of the wavelength peak occurred in transmission spectra.
2.2. Nanosensor device and electrophoresis-enhanced LSPR system The preparation process of nanocup array involves a series of steps including gold nanoparticles fabrication by electron beam lithography and nanocup production by nanoimprint [12,13]. The nanocone template on plastic substrate was passivated with dimethyl dichlorosilane solution for 30 min, followed by rinsing in ethanol. A 250 m thick flexible (Poly)ethylene terephthalate (PET) was used as supporting substrate, and a Teflon roller was used for evenly distributing the UV curable polymer on the template and PET interface. In order to cure the UV polymer, a UV light-curing flood lamp system (EC-Series, Dymax, USA) with average power density of 105 mW/cm2 was applied for 60 s at room temperature. For the Au evaporation a six pocket e-beam evaporation system (FC/BJD2000, Temescal, USA) was used. A thin adhesive layer of titanium (5 nm) was deposited before the evaporation of gold nanoparticle film (50 nm). Fig. 1C showed the photograph of the nanocup array image of scanning electron microscope (SEM, XL30-ESEM, Phlips, Netherland), indicating a periodic and uniform cup array. The optical device included a halogen cold light source (DTMINI-2, Ocean Optics Inc., Dunodin, USA) and a spectrophotometer (USB2000+, Ocean Optics Inc., Dunodin, USA), which were connected to the light emitting probe and the receptor probe respectively with fiber bundles. The nanosensor was placed on the bottom of the transparent reacting chamber. The emitting probe and receptor probe were fixed respectively below and above the reacting chamber, with 1 cm away from metal frame. With the calibration of collimating lens, the emitting light was kept vertical against the electrodes, delivered through the nanosensor and ITO electrode, finally received by the CCD. During the optical measurement, response events were monitored by tracking the resonance wavelength shifts in optical transmission spectra. The range of transmission spectra was from 300 nm to 700 nm with 0.38 nm interval. The transmission spectra were recorded and saved every 30 s simultaneously. While for electrophoresis-enhanced measurement, in order to confirm the optimal voltage, voltages varied from 0.5 V to 5 V were applied between the positive and negative electrodes. We observed the LSPR peak shifts in the transmission spectra and characterized the peak position induced by the surface potential variation. Finally, the optimal voltage was selected for the following thrombin detection by analyzing the statistical results of the peak shifts. 2.3. Synthesis and immobilization of the thrombin specific peptide The optimal thrombin cleavage site is A-B-Pro-Arg--X-Y (A and B are hydrophobic amino acids, X and Y are non-acidic amino acids). The common sequence is Leu-Val-Pro-Arg--Gly-Ser [24,25]. The peptide (Cys-Leu-Val-Pro-Arg-Gly-Ser-Cys) with the thrombin specific cleavage site was chemically synthesized by solid phase method, which was later used as the bio-recognition component for thrombin detection. Cysteine containing both thiol ( SH) and amino ( NH2 ) functional groups was added at both ends of the thrombin specific cleavage site, generating a covalent bond of AuS on the surface of the nanosensor. The chemical synthesis of the peptide was performed with solid phase method, which basically is the sequential adding of amino acids to the growing chain to obtain the peptide. High performance liquid chromatography (HPLC) and mass spectrometry (MS) were then operated to test the properties of the synthesized peptides. The peptides were stored in the form of freeze-dried powders before use. A sandwich structure was designed for the better performance of the nanosensor. The specially designed polyethylene glycol (MW 2100 Da, SH-PEG-COOH in short) was widely used to produce a
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Fig. 1. Schematic of the experimental apparatus. (A) Optical and electrical detection system with ITO electrode and the nanosensor. (B) The reaction mechanism of thrombin to the peptide. (C) SEM of the nanocup array with diameters of ∼200 nm.
robust membrane for molecular binding [26]. Negative electric charged BSA was used to increase signals in the assays. For the immobilization of the PEG, peptide and BSA, the nanosensor was thoroughly rinsed with ethanol and ultrapure water respectively to remove any organic residues off the substrate. Subsequently, the HS-PEG-COOH solution was added to the nanosensor device for the formation of stable Au-S covalent bonds, which could offer a membrane structure for bio-modification. After approximately 24 h, unbound HS-PEG-COOH were washed away with ultrapure water. Immobilization of the peptide to the PEG was then achieved by linking the amine functional groups on the peptide to the carboxyl groups of PEG through amide linkages in the presence of catalyst, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The unreacted EDC/NHS solutions and unbound peptides were rinsed off with PBS after 4 h incubation. The immobilization of BSA on the peptide was achieved with the same method. In order to demonstrate the immobilization process, potentiodynamic electrochemical measurement of cyclic voltammetry was performed on a CHI660E electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China) with a conventional three-electrode system. The nanosensor was used as working electrode, while platinum electrode and Ag/AgCl electrode were used as counter-electrode and reference electrode respectively. Cyclic voltammetry scanning was carried out on the modified nanosensor during −0.2 V–0.6 V, with scanning rate of 0.02 V/s. K4 [Fe(CN)6 ]/K3 [Fe(CN)6 ] (1:1) of 5 mM was employed as redox couple in aqueous solution to generate redox current for electrochemical properties analysis.
2.4. Detection of the thrombin Before measurement the nanosensor device was firstly immobilized with a sandwich structure. The transmission spectrum under this condition was chosen as the control group. The thrombin was prepared in phosphate buffer solution (PBS, pH = 7.4) with 1 mg/mL and stored at 4 ◦ C. In the direct detection for thrombin at different concentrations, thrombin solution was diluted to 10−11 M, 10−10 M, 10−9 M, 10−8 M, and 10−7 M with PBS. With the electrophoresis-enhanced LSPR measurement, the relationship between the concentration of thrombin and the wavelength shifts
in the transmission spectra was established. In further experiment, human albumin solution (HSA) and BSA were used as the control groups to detect the specificity of the biosensor. The experiments were repeated three times for each concentration in order to verify the results. Reagents in the experiments were all purchased from SigmaAldrich (USA). All of the above experiments were performed at room temperature (22 ◦ C). 3. Results and discussion 3.1. The thrombin-specific peptide synthesis Peptides were considered as suitable agents in the biological surface designs for sensing, which have drawn much attention due to their diverse functionality and molecular recognition abilities [27–29]. Generally, a peptide-based self-assembly with lower production cost and better stability made it a powerful tool for the development of biosensors. Due to the advanced technology of the chemical synthesis, the synthetic peptides with high purity could be well applied to specific detections. The peptide containing the thrombin specific cleavage of Arg-Gly was chemically synthesized for thrombin detection. Highperformance liquid chromatography (HPLC) and mass spectrum (MS) were used to measure the purity and check the sequence of the thrombin specific peptide. As shown in Fig. 2A, the retention time was 8.899 min with a significant peak, and the concentration of the peptide was about 95.8% by calculating the peak area, which indicated the synthesized peptide with a high purity of 95.8%. As shown in Fig. 2B, the MS test result showed a pulse at 834.82 for [M + H]+ , suggesting the peptide’s molecular mass was 833.82 in accord with the computed result based on the amino acid sequences (Cys-LeuVal-Pro-Arg-Gly-Ser-Cys). Thus, the HPLC and MS results illustrated that the synthesized peptide sequence possessing high purity and could be used for the modification of the nanosensor for thrombin detection. 3.2. Characterization of the modified nanosensor PEG is one of the most widely used inert and biocompatible linkers in surface engineering [30,31]. It could form a densely
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The current peaks decreased from 0.18 mA/cm2 to 0.05 mA/cm2 and the redox peaks were all at 0.16 V and 0.28 V. The decreasing current attributed to the increasing impedance, which indicated modification of the nanosensor was successful. Moreover, the same characteristic redox potentials and almost symmetric shapes of the cyclic voltammetry illustrated that the nanostructure device had a relatively good stability. The presence of PEG on the nanosensor surface provided a robust membrane for peptide immobilization. When peptides were immobilized by PEG, they were all arranged through amido bonds, which could facilitate the reactions between thrombin and the peptides. BSA is a kind of protein with electronegativity, which was often used to stabilize enzymes and to prevent adhesion of the enzyme to reaction tubes, pipet tips, and other vessels. Theoretically, immobilization of the sandwich structure with PEG, peptide, and BSA could provide an excellent sensing platform for thrombin detection.
3.3. Electrophoresis enhancement on LSPR
Fig. 2. Qualification of the thrombin-specific peptide. (A) The high-performance liquid chromatography (HPLC) test for the peptide. (B) The mass spectrum (MS) test for the peptide.
Fig. 3. Cyclic voltammetry of nanocup array (NanoCA), NanoCA + PEG, NanoCA + PEG + peptide, and NanoCA + PEG + peptide + BSA (scope: −0.2 V–0.6 V, scan rate: 0.02 V/s).
packed self-assembled monolayer on gold electrodes and comprise a uniform morphology with the ethylene glycol units in a helical conformation, which were predominantly oriented perpendicular to the surfaces [32,33]. With the immobilization of the PEG, peptide, and BSA on the nanosensor surface, corresponding change of interfacial properties occurred. The characterizations of the electrode surface were performed by cyclic voltammetry. As shown in Fig. 3, with the immobilization of PEG, peptide, and BSA successively, the height of the current peak decreased gradually, whilst the anodic and cathodic peak potential shared the same magnitude.
As indicated previously, thrombin would bind to specific cleavage site, followed by cleaving the composite. Finally, BSA and partial peptides would be released from the nanosensor surface in the absence of chemical bonds. However, these released proteins and peptides around nanocup array made the effective spectral signals difficult to be detected and further reduce the detection sensitivity. Electrophoresis can directionally guide the charged molecules towards or away from the sensor surface, which was used as the amplification in this LSPR detection. The LSPR response was measured in the optical transmission spectrum. There were three peaks in the transmission spectra. The first peak at around 520 nm and the third peak at around 600 nm were caused by Wood’s anomaly (WA), which were unstable and varied greatly between batches. The second peak at around 570 nm was caused by Mie scattering, which were decided by the gold nanoparticles [13]. In our experiment, the thrombin specific peptide was modified on the gold nanoparticles. So we selected the second peak as the major variable of observation. In our study, the nanosensor device was immobilized with a sandwich structure including thrombin specific peptide. Due to the cleavage of the peptide, the refractive index on the sensor surface decreased. Therefore, the decreased refractive index would induce blue shift in the peaks. As shown in Fig. 4A, the LSPR peak in the transmission spectra at the range from 550 nm to 580 nm shifted to the left when thrombin was added to the reactor, and when direct current was added to the nanosensor, the offset enhanced. The phenomenon illustrated that the direct current could result in significant LSPR peak shifts. With direct current applied, the negatively charged BSA was freed from the sensing surface, which induced the shift of the resonance peak in transmission spectra. Besides, we could find that the incubation time of thrombin was about 8 min. The thrombin detection time was about 20 min. So, the time in further measurement was fixed at 20 min. As shown in Fig. 4B, the entire transmission spectra had violetshift with different voltages from 0.5 V to 5 V. The shift of LSPR peak increased as the direct current applied between the electrodes increasing. The resonance wavelength shifts with 3 V demonstrated a high sensitivity of nanosensor for refractive index changes. When the direct current increased to 5 V, the fluids in the reaction chamber was transformed into epinephelos state whilst decrease in transmission shift was observed, which might result from the dissociation of the molecules from the sensor surface and the oxidation of the gold film. Therefore, the shifts in transmission spectrum could be used as an indicator for analyzing refractive index changes of the nanosensor to the analyte.
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Fig. 4. The enhancement effect of direct current (DC) on LSPR measurement. (A) The wavelength shifts with time. (B) The transmission spectrograms with 0 V, 0.5 V, 1 V, 3 V, and 5 V. Inset: the statistics of the resonance wavelength shifts (every measurement repeated for 3 times).
3.4. Thrombin detection with the electrophoresis-enhanced LSPR By applying the technology of electrophoresis enhancement in LSPR on the nanosensor, the biosensor can be modified with a higher sensitivity in the measurement. Based on the modification of thrombin-specific peptide on the nanosensor and the enhancement of the electrophoresis on LSPR, the increasing concentration of thrombin fostered increasing refractive index changes on the sensor surface, which would lead to the increasing LSPR peak shifts. The reflection spectra obtained from the nanosensor are shown in Fig. 5A. With the introduction of thrombin, the resonance wavelength of the peak was only shifted from 574.2 nm to 570.5 nm. Whilst with the effect of direct current, the peak resonance wavelength was shifted from 574.2 nm to 563.8 nm. Fig. A.1 and Fig. A.2 showed wavelength shifts in transmission spectra under different concentrations (10−11 M, 10−10 M, 10−9 M, 10−8 M, and 10−7 M) with DC and without DC, respectively. Thus, the LSPR peak shifts were elicited by the reaction of thrombin and strengthened by direct current from 3.7 nm to 10.4 nm, indicating that the direct current on the nanosensor surface enhanced the wavelength shifts in LSPR. As shown in Fig. 5B, the wavelength shifts in transmission spectrum were presented when various concentrations of thrombin (10−11 M, 10−10 M, 10−9 M, 10−8 M, and 10−7 M) were respectively introduced. Through analysis, data obtained from LSPR responses in peak shifts demonstrated a linear relationship with log concentrations of thrombin. Under the same concentration of thrombin, direct current generated more significant peak violet shifts in comparison with the sole LSPR measurement. In addition, among different concentrations, LSPR with direct current showed a significant sensitivity and a well linearity towards thrombin detection. Thereof the electrophoresis-enhanced LSPR sensing could be developed into a sensitive analyte technique that to some extent overcome diffusion efficiency and push the limit of detection beyond the scope of the conventional LSPR sensors. In order to validate the specificity of the biosensor, BSA and HSA were further studied. As shown in Fig. 6A, the thrombin had the significant wave peak left shift in the transmission spectrum, while the wave peak of BSA and HSA almost had no changes. The analytical results in Fig. 6B also indicated that the peak shifts between thrombin and BSA, HSA had significant differences with *P < 0.0001, implying that the biosensor with the modification of specific thrombin peptide maintained the selectivity to the thrombin. Thus, with specific binding of biomolecules on the sensor surface, the electrophoresis-enhanced LSPR measurement could be designed for various selective analytes.
Plasmonic nanostructures have played a significant role in the field of nanotechnology, which renders them precious for various sensing applications such as LSPR, colorimetric sensing, and surface enhanced Raman scattering (SERS) [8,34]. Among them, LSPR-based sensing with high sensitivity and reproducibility has been extensively utilized in biological and chemical analysis. Several methods have been employed to boost the sensitivity of LSPR including well-designed nanostructure devices with improved refractive index sensitivity, and applying electric field to promote the molecular diffusion [5,16,35]. Due to the unprecedented high sensitivity (about 46000 nm per RIU), nanocup devices were very sensitive in detecting small changes in refractive index of the analyte and gave rise to a huge shift in the resonance wavelength [12,13]. In our experiments, the electrophoresis enhanced LSPR on nanocup array showed greater response and sensitivity than that of sole LSPR measurement. Although the combining of the electrophoresis and LSPR seems to be little complicated, the LSPR signals were enhanced in extra peak shift by direct current greatly. What’s more, the gold nanoparticles inside the nanosensor possessed excellent electrocatalysis abilities for various chemicals and biomolecules, which provided a promising method for the biosensor design. In addition, low-cost flexible substrates for nanoplasmonic sensing have played a significant role in the field of LSPR [34,36]. In our study, the nanocup array was fabricated on a plastic substrate using nanoimprint lithography technology. With this technology, the fabrication process is simplified and the overall cost is low. Therefore, the combination of electrophoresis and LSPR on nanocup array provided a relatively low-cost method toward real-world applications in bio-detection. Aptamers are specific DNA or RNA strands obtained from random-sequence nucleic acid libraries. They could selectively bind to proteins or target molecules with high affinities. Most of the thrombin biosensors utilized the known sequence of thrombin aptamer as probes to detect thrombin [37–39]. However, the incubation and detection time could take as long as several hours. The specific peptide could be cleaved by thrombin, which can determine catalytic activity and shorten the reaction time. What’s more, peptides have several unique features, such as synthesis convenience, ease of chemical modification, and structural stability and flexibility, which make them an idea alternative material for development of biosensors. In our study, the sandwich structure including thrombin specific peptide had a high sensitivity of thrombin on the electrophoresis-enhanced LSPR. Considering the thrombin detection time showed in Table 1, it may imply that using electrophoresis can contribute to shortening measurement time
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Fig. 5. Electrophoresis-enhanced LSPR sensor for thrombin detection. (A) The transmission spectrograms of nanosensor with DC and without DC. (B) The statistics of the wavelength shifts from 10−11 M to 10−7 M thrombin detected by the peak shifts (every measurement repeated for 3 times).
Fig. 6. Selectivity of the nanosensor. (A) The transmission spectrograms with thrombin, BSA, and HSA. (B) Statistics for wavelength shifts with thrombin, HSA, and BSA with 10−7 M (every measurement repeated for 3 times).
Table 1 Comparison of different thrombin biosensors. Technique
Probe unit
Detection time
Limit of detection
Reference
Fluorescence Fluorescence LSPR Electrochemical Electrochemical Photoelectrochemical Electrophoresis-enhanced LSPR
DNA aptamer RNA aptamer Aptamer-AuNPs/Fe3 O4 DNA aptamer Peptide Peptide Peptide
2h 2h 30 min 50 min 30 min 60 min 20 min
0.03 nM 10 fM 200 pM 5 pg/mL 28 fM 1.9 ug/mL 10 pM
[40] [41] [42] [43] [24] [44] The present study
to some degrees. Besides, the normal concentration of thrombin detection in real sample was from 100 pM to 2.8 nM. Our detection scope was from 10 pM to 100 nM, which encompassed the target concentration of thrombin. Furthermore, with specific binding of biomolecules, the nanosensor applied on the electrophoresisenhanced LSPR would be holding great potential to be used as a simple, sensitive, and selective platform for the chemical and biological detection. The nanocup array sensor decorated by peptides containing the thrombin-specific cleavage site was used to construct an electrophoresis-enhanced LSPR biosensor. Modified by a sandwich structure of PEG, peptide, and BSA, the nanosensor showed a significant sensitivity and specificity in the detection of thrombin. Through combining the nanosensor with specific receptors such as peptide to the analyte, the electrophoresis-enhanced LSPR would allow for application in other biochemical sensing.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81371643, 81102705), the Zhejiang Provincial Natural Science Foundation of China for Distinguished Young Scholars (Grant No. LR13H180002), theacademic leader of the academic climbing project of undergraduate colleges and universities in the Zhejiang province (Grant No. Pb2013211).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.03.134.
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Biographies Shuang Li received her bachelor degree from Hunan normal University in 2014. Now she is an M.Sc student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Diming Zhang received his bachelor degree from Zhejiang University, in 2011. Now he is a Ph.D. student of biomedical engineering of Zhejiang University. His work includes design of nanoplasmonic biosensor and electronic measurement. Qian Zhang received her bachelor degree from Zhejiang University in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Yanli Lu received her bachelor degree from Xi’an Jiaotong University, PR China in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes based biosensors and electronic measurement. Nantao Li will receive his bachelor degree from Zhejiang University in 2016. Currently he is pursuing a graduate project for further study. His work includes design of nanoplasmonic sensor and medical imaging. Qunwei Chen received his MS degree and MD degree from Zhejiang Chinese Medical University and Shanghai Chinese Medical University, PR China, respectively. He is currently in Zhejiang Chinese Medical University. His research is mainly focused on cancer. Qingjun Liu received his Ph.D. degree in biomedical engineering from Zhejiang University, PR China in 2006. He is currently a professor in Biosensor National Special Laboratory, Zhejiang University. He is also a visiting scholar in the Micro and Nanotechnology Laboratory (MNTL) at the University of Illinois at Urbana-Champaign (UIUC). He published the book of Cell-Based Biosensors: Principles and Applications, by Artech House Publishers USA in October 2009. His research interests concentrate on the biosensors (e.g. living cell sensor, DNA sensor and protein sensor) and BioMEMS system.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrophoresis-enhanced localized surface plasmon resonance sensing based on nanocup array for thrombin detection Shuang Li a,b , Diming Zhang a , Qian Zhang a , Yanli Lu a , Nantao Li a , Qunwei Chen c , Qingjun Liu a,b,∗ a Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, 310027, PR China b Collaborative Innovation Center of Health Management, Fujian University of Traditional Chinese Medicine, Fuzhou, 350108, PR China c Zhejiang Chinese Medical University, 310053, PR China
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Article history: Received 25 December 2015 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online 25 March 2016 Keywords: Localized surface plasmon resonance (LSPR) Electrophoresis Nanocup array Thrombin Biosensor
a b s t r a c t Based on the special electrical and optical properties resulted from periodic nanostructure, localized surface plasmon resonance (LSPR) sensors have been widely used for various chemical and biological detections. Utilizing uniform alignment nanocups, an electrophoresis-enhanced LSPR sensor was designed for sensitive thrombin detection. The nanocup array was composed of nano-scaled funnel shaped cups with nanoparticles along the side walls. Through self-assembly, polyethylene glycol, thrombin-specific peptide, and bovine serum albumin were successively immobilized on the nanocup array. The results demonstrated that the synchronous implementation of electrophoresis and LSPR measurement could lead to obvious peak shifts in optical transmission spectra for detecting thrombin. The detection limit was as low as 10−11 M. With the high sensitivity and well linearity, the electrophoresisenhanced LSPR sensing offered a novel design perspective for chemical and biological sensors with specific modifications on the nanosensor surfaces. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Recent progresses in highly sensitive optical transducers, which combined with the exquisite specificity, affinity, and versatility of bio-molecular interactions, have greatly promoted the development of a wide variety of optical biosensors in the field of biomedical and environmental science. Among these sensors, localized surface plasmon resonance (LSPR) was one of the most widely utilized techniques in optical biosensors design to measure molecular binding kinetics by the virtue of density oscillations of conduction electrons on the metallic nanostructure surfaces, which varied with the affinity of the analytes [1–3]. The mechanism of these LSPR sensors was often based on the changes of transmission spectra, which responding to refractive index changes from the binding of molecules on the nanoparticle surfaces. Typically, the increase of refractive index would lead to the red shift. While, the decrease of refractive index would lead to the blue shift in spectral absorption and scattering peaks [4–8].
∗ Corresponding author. E-mail address: [email protected] (Q. Liu). http://dx.doi.org/10.1016/j.snb.2016.03.134 0925-4005/© 2016 Elsevier B.V. All rights reserved.
LSPR phenomenon on nanostructured devices (e.g. nanoparticles, nanoholes, and nanorods) had high sensitivities in detecting small changes in refractive index of analytes. However, the difficulties in the size control of the nanoparticles and the multiple scattering peaks of the nanoholes were often the obstacles on the LSPR detection. Lycurgus cup can appear different colors under the condition of different light illumination directions. According to some publications, it was due to metal nanoparticle optical scattering and inspired the research in the field of nanoplasmonics [9–11]. Giving rise to the effect of the Lycurgus cup structure, nanocup array is characterized by transmission peaks in the visible wavelength and has a high sensitivity in detecting small changes in refractive index of analyte. Some groups have reported that the hybrid structures of nanocup array with nanoparticles have a more stable optical performance in transmission spectra, which could be used to improve stabilities of the LSPR detections [12,13]. On the other hand, diffusion of the target molecules was also often limited during the mass transportation process on the plasmon resonance surface, which could slow down the molecular binding velocity and thereof limit the sensitivity of sensors. In order to improve the efficiency of mass transportation, electrokinetic preconcentration of charged molecules toward sensing surface could accelerate the analytes binding, which have been studied by several
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groups [14–16]. At the same time, the intensity of LSPR could also be influenced by electron densities and electrical attractions in the electrochemical scanning, which could enhance the LSPR signals [17]. The electrochemical scanning could change redox reaction conditions around the nanosensor surface, elicit refractive index changes, finally generate extra LSPR wavelength shifts [13]. Therefore, the combination of electrophoresis and LSPR may provide a promising approach to enhance LSPR signals with significant resonance wavelength shifts in transmission spectra for analyte detections. Thrombin is a serine protease that plays an important role in blood coagulation, thrombosis, inflammation, angiogenesis, tumor growth and metastasis [18,19]. Various sensing methods have been reported for the detection of thrombin, such as quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and electrochemical analyses, which were mostly on the basis of thrombin aptamers [20,21]. In order to improve the specificity of the detection, self-assembled peptides have been considering as novel approaches for modification of sensors in biological detection [22,23]. Without the redundant structures, peptides could result in the capability for practical applications through simplifying the manufacture. Thus, the thrombin specific cleavage peptides have been used to fabricate ultrasensitive thrombin biosensors to study the mechanistic details of cleavage processes [24]. In this paper, an electrophoresis-enhanced LSPR measuring method based on peptide modified nanocup array was used as the nanosensor for thrombin detection. The surface of the nanocups was modified with a sandwich structure including the polyethylene glycol (PEG), thrombin specific peptide, and bovine serum albumin (BSA). By applying direct current onto the electrodes, the transport of molecules toward the sensor surfaces was accelerated and the resonance wavelength shifts in the transmission spectra were enhanced significantly. The electrophoresis-enhanced LSPR sensing method might provide a promising approach for sensitive and selective thrombin detection.
2. Experiments 2.1. Electrophoresis-enhanced LSPR for thrombin detection The schematic of the experimental setup is shown in Fig. 1A. The halogen cold laser was delivered through the nanosensor and indium tin oxide (ITO) electrode, finally received by a CCD camera. The direct current was applied between the nanosensor and ITO electrodes, which were utilized as the positive and negative electrodes, respectively, for electrophoresis enhanced LSPR measurement. In the measurement, the gap distance between the ITO electrode and the nanosensor was fixed at 1 cm. As shown in Fig. 1B, a “sandwich” strategy was employed. Polyethylene glycol (PEG) used to form a membrane on the nanosurface for better immobilization of the peptide. The thrombin specific peptide was used to determine the thrombin catalytic activity. The negative charged bovine serum albumin (BSA) was added at the terminus of the peptides to amplify the LSPR signals by electrophoresis, for which could accelerate the peptide’s cleavage reaction. The thrombin solution was introduced into the nanosensor surface, which was followed by thrombin cleavage at its specific site. With direct current applied between the ITO electrode and the nanosensor surface, the cleaved peptide and electronegative BSA would be separated. In the meantime, due to the refractive index changes on the sensor surface, significant violet-shift of the wavelength peak occurred in transmission spectra.
2.2. Nanosensor device and electrophoresis-enhanced LSPR system The preparation process of nanocup array involves a series of steps including gold nanoparticles fabrication by electron beam lithography and nanocup production by nanoimprint [12,13]. The nanocone template on plastic substrate was passivated with dimethyl dichlorosilane solution for 30 min, followed by rinsing in ethanol. A 250 m thick flexible (Poly)ethylene terephthalate (PET) was used as supporting substrate, and a Teflon roller was used for evenly distributing the UV curable polymer on the template and PET interface. In order to cure the UV polymer, a UV light-curing flood lamp system (EC-Series, Dymax, USA) with average power density of 105 mW/cm2 was applied for 60 s at room temperature. For the Au evaporation a six pocket e-beam evaporation system (FC/BJD2000, Temescal, USA) was used. A thin adhesive layer of titanium (5 nm) was deposited before the evaporation of gold nanoparticle film (50 nm). Fig. 1C showed the photograph of the nanocup array image of scanning electron microscope (SEM, XL30-ESEM, Phlips, Netherland), indicating a periodic and uniform cup array. The optical device included a halogen cold light source (DTMINI-2, Ocean Optics Inc., Dunodin, USA) and a spectrophotometer (USB2000+, Ocean Optics Inc., Dunodin, USA), which were connected to the light emitting probe and the receptor probe respectively with fiber bundles. The nanosensor was placed on the bottom of the transparent reacting chamber. The emitting probe and receptor probe were fixed respectively below and above the reacting chamber, with 1 cm away from metal frame. With the calibration of collimating lens, the emitting light was kept vertical against the electrodes, delivered through the nanosensor and ITO electrode, finally received by the CCD. During the optical measurement, response events were monitored by tracking the resonance wavelength shifts in optical transmission spectra. The range of transmission spectra was from 300 nm to 700 nm with 0.38 nm interval. The transmission spectra were recorded and saved every 30 s simultaneously. While for electrophoresis-enhanced measurement, in order to confirm the optimal voltage, voltages varied from 0.5 V to 5 V were applied between the positive and negative electrodes. We observed the LSPR peak shifts in the transmission spectra and characterized the peak position induced by the surface potential variation. Finally, the optimal voltage was selected for the following thrombin detection by analyzing the statistical results of the peak shifts. 2.3. Synthesis and immobilization of the thrombin specific peptide The optimal thrombin cleavage site is A-B-Pro-Arg--X-Y (A and B are hydrophobic amino acids, X and Y are non-acidic amino acids). The common sequence is Leu-Val-Pro-Arg--Gly-Ser [24,25]. The peptide (Cys-Leu-Val-Pro-Arg-Gly-Ser-Cys) with the thrombin specific cleavage site was chemically synthesized by solid phase method, which was later used as the bio-recognition component for thrombin detection. Cysteine containing both thiol ( SH) and amino ( NH2 ) functional groups was added at both ends of the thrombin specific cleavage site, generating a covalent bond of AuS on the surface of the nanosensor. The chemical synthesis of the peptide was performed with solid phase method, which basically is the sequential adding of amino acids to the growing chain to obtain the peptide. High performance liquid chromatography (HPLC) and mass spectrometry (MS) were then operated to test the properties of the synthesized peptides. The peptides were stored in the form of freeze-dried powders before use. A sandwich structure was designed for the better performance of the nanosensor. The specially designed polyethylene glycol (MW 2100 Da, SH-PEG-COOH in short) was widely used to produce a
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Fig. 1. Schematic of the experimental apparatus. (A) Optical and electrical detection system with ITO electrode and the nanosensor. (B) The reaction mechanism of thrombin to the peptide. (C) SEM of the nanocup array with diameters of ∼200 nm.
robust membrane for molecular binding [26]. Negative electric charged BSA was used to increase signals in the assays. For the immobilization of the PEG, peptide and BSA, the nanosensor was thoroughly rinsed with ethanol and ultrapure water respectively to remove any organic residues off the substrate. Subsequently, the HS-PEG-COOH solution was added to the nanosensor device for the formation of stable Au-S covalent bonds, which could offer a membrane structure for bio-modification. After approximately 24 h, unbound HS-PEG-COOH were washed away with ultrapure water. Immobilization of the peptide to the PEG was then achieved by linking the amine functional groups on the peptide to the carboxyl groups of PEG through amide linkages in the presence of catalyst, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The unreacted EDC/NHS solutions and unbound peptides were rinsed off with PBS after 4 h incubation. The immobilization of BSA on the peptide was achieved with the same method. In order to demonstrate the immobilization process, potentiodynamic electrochemical measurement of cyclic voltammetry was performed on a CHI660E electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China) with a conventional three-electrode system. The nanosensor was used as working electrode, while platinum electrode and Ag/AgCl electrode were used as counter-electrode and reference electrode respectively. Cyclic voltammetry scanning was carried out on the modified nanosensor during −0.2 V–0.6 V, with scanning rate of 0.02 V/s. K4 [Fe(CN)6 ]/K3 [Fe(CN)6 ] (1:1) of 5 mM was employed as redox couple in aqueous solution to generate redox current for electrochemical properties analysis.
2.4. Detection of the thrombin Before measurement the nanosensor device was firstly immobilized with a sandwich structure. The transmission spectrum under this condition was chosen as the control group. The thrombin was prepared in phosphate buffer solution (PBS, pH = 7.4) with 1 mg/mL and stored at 4 ◦ C. In the direct detection for thrombin at different concentrations, thrombin solution was diluted to 10−11 M, 10−10 M, 10−9 M, 10−8 M, and 10−7 M with PBS. With the electrophoresis-enhanced LSPR measurement, the relationship between the concentration of thrombin and the wavelength shifts
in the transmission spectra was established. In further experiment, human albumin solution (HSA) and BSA were used as the control groups to detect the specificity of the biosensor. The experiments were repeated three times for each concentration in order to verify the results. Reagents in the experiments were all purchased from SigmaAldrich (USA). All of the above experiments were performed at room temperature (22 ◦ C). 3. Results and discussion 3.1. The thrombin-specific peptide synthesis Peptides were considered as suitable agents in the biological surface designs for sensing, which have drawn much attention due to their diverse functionality and molecular recognition abilities [27–29]. Generally, a peptide-based self-assembly with lower production cost and better stability made it a powerful tool for the development of biosensors. Due to the advanced technology of the chemical synthesis, the synthetic peptides with high purity could be well applied to specific detections. The peptide containing the thrombin specific cleavage of Arg-Gly was chemically synthesized for thrombin detection. Highperformance liquid chromatography (HPLC) and mass spectrum (MS) were used to measure the purity and check the sequence of the thrombin specific peptide. As shown in Fig. 2A, the retention time was 8.899 min with a significant peak, and the concentration of the peptide was about 95.8% by calculating the peak area, which indicated the synthesized peptide with a high purity of 95.8%. As shown in Fig. 2B, the MS test result showed a pulse at 834.82 for [M + H]+ , suggesting the peptide’s molecular mass was 833.82 in accord with the computed result based on the amino acid sequences (Cys-LeuVal-Pro-Arg-Gly-Ser-Cys). Thus, the HPLC and MS results illustrated that the synthesized peptide sequence possessing high purity and could be used for the modification of the nanosensor for thrombin detection. 3.2. Characterization of the modified nanosensor PEG is one of the most widely used inert and biocompatible linkers in surface engineering [30,31]. It could form a densely
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The current peaks decreased from 0.18 mA/cm2 to 0.05 mA/cm2 and the redox peaks were all at 0.16 V and 0.28 V. The decreasing current attributed to the increasing impedance, which indicated modification of the nanosensor was successful. Moreover, the same characteristic redox potentials and almost symmetric shapes of the cyclic voltammetry illustrated that the nanostructure device had a relatively good stability. The presence of PEG on the nanosensor surface provided a robust membrane for peptide immobilization. When peptides were immobilized by PEG, they were all arranged through amido bonds, which could facilitate the reactions between thrombin and the peptides. BSA is a kind of protein with electronegativity, which was often used to stabilize enzymes and to prevent adhesion of the enzyme to reaction tubes, pipet tips, and other vessels. Theoretically, immobilization of the sandwich structure with PEG, peptide, and BSA could provide an excellent sensing platform for thrombin detection.
3.3. Electrophoresis enhancement on LSPR
Fig. 2. Qualification of the thrombin-specific peptide. (A) The high-performance liquid chromatography (HPLC) test for the peptide. (B) The mass spectrum (MS) test for the peptide.
Fig. 3. Cyclic voltammetry of nanocup array (NanoCA), NanoCA + PEG, NanoCA + PEG + peptide, and NanoCA + PEG + peptide + BSA (scope: −0.2 V–0.6 V, scan rate: 0.02 V/s).
packed self-assembled monolayer on gold electrodes and comprise a uniform morphology with the ethylene glycol units in a helical conformation, which were predominantly oriented perpendicular to the surfaces [32,33]. With the immobilization of the PEG, peptide, and BSA on the nanosensor surface, corresponding change of interfacial properties occurred. The characterizations of the electrode surface were performed by cyclic voltammetry. As shown in Fig. 3, with the immobilization of PEG, peptide, and BSA successively, the height of the current peak decreased gradually, whilst the anodic and cathodic peak potential shared the same magnitude.
As indicated previously, thrombin would bind to specific cleavage site, followed by cleaving the composite. Finally, BSA and partial peptides would be released from the nanosensor surface in the absence of chemical bonds. However, these released proteins and peptides around nanocup array made the effective spectral signals difficult to be detected and further reduce the detection sensitivity. Electrophoresis can directionally guide the charged molecules towards or away from the sensor surface, which was used as the amplification in this LSPR detection. The LSPR response was measured in the optical transmission spectrum. There were three peaks in the transmission spectra. The first peak at around 520 nm and the third peak at around 600 nm were caused by Wood’s anomaly (WA), which were unstable and varied greatly between batches. The second peak at around 570 nm was caused by Mie scattering, which were decided by the gold nanoparticles [13]. In our experiment, the thrombin specific peptide was modified on the gold nanoparticles. So we selected the second peak as the major variable of observation. In our study, the nanosensor device was immobilized with a sandwich structure including thrombin specific peptide. Due to the cleavage of the peptide, the refractive index on the sensor surface decreased. Therefore, the decreased refractive index would induce blue shift in the peaks. As shown in Fig. 4A, the LSPR peak in the transmission spectra at the range from 550 nm to 580 nm shifted to the left when thrombin was added to the reactor, and when direct current was added to the nanosensor, the offset enhanced. The phenomenon illustrated that the direct current could result in significant LSPR peak shifts. With direct current applied, the negatively charged BSA was freed from the sensing surface, which induced the shift of the resonance peak in transmission spectra. Besides, we could find that the incubation time of thrombin was about 8 min. The thrombin detection time was about 20 min. So, the time in further measurement was fixed at 20 min. As shown in Fig. 4B, the entire transmission spectra had violetshift with different voltages from 0.5 V to 5 V. The shift of LSPR peak increased as the direct current applied between the electrodes increasing. The resonance wavelength shifts with 3 V demonstrated a high sensitivity of nanosensor for refractive index changes. When the direct current increased to 5 V, the fluids in the reaction chamber was transformed into epinephelos state whilst decrease in transmission shift was observed, which might result from the dissociation of the molecules from the sensor surface and the oxidation of the gold film. Therefore, the shifts in transmission spectrum could be used as an indicator for analyzing refractive index changes of the nanosensor to the analyte.
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Fig. 4. The enhancement effect of direct current (DC) on LSPR measurement. (A) The wavelength shifts with time. (B) The transmission spectrograms with 0 V, 0.5 V, 1 V, 3 V, and 5 V. Inset: the statistics of the resonance wavelength shifts (every measurement repeated for 3 times).
3.4. Thrombin detection with the electrophoresis-enhanced LSPR By applying the technology of electrophoresis enhancement in LSPR on the nanosensor, the biosensor can be modified with a higher sensitivity in the measurement. Based on the modification of thrombin-specific peptide on the nanosensor and the enhancement of the electrophoresis on LSPR, the increasing concentration of thrombin fostered increasing refractive index changes on the sensor surface, which would lead to the increasing LSPR peak shifts. The reflection spectra obtained from the nanosensor are shown in Fig. 5A. With the introduction of thrombin, the resonance wavelength of the peak was only shifted from 574.2 nm to 570.5 nm. Whilst with the effect of direct current, the peak resonance wavelength was shifted from 574.2 nm to 563.8 nm. Fig. A.1 and Fig. A.2 showed wavelength shifts in transmission spectra under different concentrations (10−11 M, 10−10 M, 10−9 M, 10−8 M, and 10−7 M) with DC and without DC, respectively. Thus, the LSPR peak shifts were elicited by the reaction of thrombin and strengthened by direct current from 3.7 nm to 10.4 nm, indicating that the direct current on the nanosensor surface enhanced the wavelength shifts in LSPR. As shown in Fig. 5B, the wavelength shifts in transmission spectrum were presented when various concentrations of thrombin (10−11 M, 10−10 M, 10−9 M, 10−8 M, and 10−7 M) were respectively introduced. Through analysis, data obtained from LSPR responses in peak shifts demonstrated a linear relationship with log concentrations of thrombin. Under the same concentration of thrombin, direct current generated more significant peak violet shifts in comparison with the sole LSPR measurement. In addition, among different concentrations, LSPR with direct current showed a significant sensitivity and a well linearity towards thrombin detection. Thereof the electrophoresis-enhanced LSPR sensing could be developed into a sensitive analyte technique that to some extent overcome diffusion efficiency and push the limit of detection beyond the scope of the conventional LSPR sensors. In order to validate the specificity of the biosensor, BSA and HSA were further studied. As shown in Fig. 6A, the thrombin had the significant wave peak left shift in the transmission spectrum, while the wave peak of BSA and HSA almost had no changes. The analytical results in Fig. 6B also indicated that the peak shifts between thrombin and BSA, HSA had significant differences with *P < 0.0001, implying that the biosensor with the modification of specific thrombin peptide maintained the selectivity to the thrombin. Thus, with specific binding of biomolecules on the sensor surface, the electrophoresis-enhanced LSPR measurement could be designed for various selective analytes.
Plasmonic nanostructures have played a significant role in the field of nanotechnology, which renders them precious for various sensing applications such as LSPR, colorimetric sensing, and surface enhanced Raman scattering (SERS) [8,34]. Among them, LSPR-based sensing with high sensitivity and reproducibility has been extensively utilized in biological and chemical analysis. Several methods have been employed to boost the sensitivity of LSPR including well-designed nanostructure devices with improved refractive index sensitivity, and applying electric field to promote the molecular diffusion [5,16,35]. Due to the unprecedented high sensitivity (about 46000 nm per RIU), nanocup devices were very sensitive in detecting small changes in refractive index of the analyte and gave rise to a huge shift in the resonance wavelength [12,13]. In our experiments, the electrophoresis enhanced LSPR on nanocup array showed greater response and sensitivity than that of sole LSPR measurement. Although the combining of the electrophoresis and LSPR seems to be little complicated, the LSPR signals were enhanced in extra peak shift by direct current greatly. What’s more, the gold nanoparticles inside the nanosensor possessed excellent electrocatalysis abilities for various chemicals and biomolecules, which provided a promising method for the biosensor design. In addition, low-cost flexible substrates for nanoplasmonic sensing have played a significant role in the field of LSPR [34,36]. In our study, the nanocup array was fabricated on a plastic substrate using nanoimprint lithography technology. With this technology, the fabrication process is simplified and the overall cost is low. Therefore, the combination of electrophoresis and LSPR on nanocup array provided a relatively low-cost method toward real-world applications in bio-detection. Aptamers are specific DNA or RNA strands obtained from random-sequence nucleic acid libraries. They could selectively bind to proteins or target molecules with high affinities. Most of the thrombin biosensors utilized the known sequence of thrombin aptamer as probes to detect thrombin [37–39]. However, the incubation and detection time could take as long as several hours. The specific peptide could be cleaved by thrombin, which can determine catalytic activity and shorten the reaction time. What’s more, peptides have several unique features, such as synthesis convenience, ease of chemical modification, and structural stability and flexibility, which make them an idea alternative material for development of biosensors. In our study, the sandwich structure including thrombin specific peptide had a high sensitivity of thrombin on the electrophoresis-enhanced LSPR. Considering the thrombin detection time showed in Table 1, it may imply that using electrophoresis can contribute to shortening measurement time
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Fig. 5. Electrophoresis-enhanced LSPR sensor for thrombin detection. (A) The transmission spectrograms of nanosensor with DC and without DC. (B) The statistics of the wavelength shifts from 10−11 M to 10−7 M thrombin detected by the peak shifts (every measurement repeated for 3 times).
Fig. 6. Selectivity of the nanosensor. (A) The transmission spectrograms with thrombin, BSA, and HSA. (B) Statistics for wavelength shifts with thrombin, HSA, and BSA with 10−7 M (every measurement repeated for 3 times).
Table 1 Comparison of different thrombin biosensors. Technique
Probe unit
Detection time
Limit of detection
Reference
Fluorescence Fluorescence LSPR Electrochemical Electrochemical Photoelectrochemical Electrophoresis-enhanced LSPR
DNA aptamer RNA aptamer Aptamer-AuNPs/Fe3 O4 DNA aptamer Peptide Peptide Peptide
2h 2h 30 min 50 min 30 min 60 min 20 min
0.03 nM 10 fM 200 pM 5 pg/mL 28 fM 1.9 ug/mL 10 pM
[40] [41] [42] [43] [24] [44] The present study
to some degrees. Besides, the normal concentration of thrombin detection in real sample was from 100 pM to 2.8 nM. Our detection scope was from 10 pM to 100 nM, which encompassed the target concentration of thrombin. Furthermore, with specific binding of biomolecules, the nanosensor applied on the electrophoresisenhanced LSPR would be holding great potential to be used as a simple, sensitive, and selective platform for the chemical and biological detection. The nanocup array sensor decorated by peptides containing the thrombin-specific cleavage site was used to construct an electrophoresis-enhanced LSPR biosensor. Modified by a sandwich structure of PEG, peptide, and BSA, the nanosensor showed a significant sensitivity and specificity in the detection of thrombin. Through combining the nanosensor with specific receptors such as peptide to the analyte, the electrophoresis-enhanced LSPR would allow for application in other biochemical sensing.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81371643, 81102705), the Zhejiang Provincial Natural Science Foundation of China for Distinguished Young Scholars (Grant No. LR13H180002), theacademic leader of the academic climbing project of undergraduate colleges and universities in the Zhejiang province (Grant No. Pb2013211).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.03.134.
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Biographies Shuang Li received her bachelor degree from Hunan normal University in 2014. Now she is an M.Sc student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Diming Zhang received his bachelor degree from Zhejiang University, in 2011. Now he is a Ph.D. student of biomedical engineering of Zhejiang University. His work includes design of nanoplasmonic biosensor and electronic measurement. Qian Zhang received her bachelor degree from Zhejiang University in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Yanli Lu received her bachelor degree from Xi’an Jiaotong University, PR China in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes based biosensors and electronic measurement. Nantao Li will receive his bachelor degree from Zhejiang University in 2016. Currently he is pursuing a graduate project for further study. His work includes design of nanoplasmonic sensor and medical imaging. Qunwei Chen received his MS degree and MD degree from Zhejiang Chinese Medical University and Shanghai Chinese Medical University, PR China, respectively. He is currently in Zhejiang Chinese Medical University. His research is mainly focused on cancer. Qingjun Liu received his Ph.D. degree in biomedical engineering from Zhejiang University, PR China in 2006. He is currently a professor in Biosensor National Special Laboratory, Zhejiang University. He is also a visiting scholar in the Micro and Nanotechnology Laboratory (MNTL) at the University of Illinois at Urbana-Champaign (UIUC). He published the book of Cell-Based Biosensors: Principles and Applications, by Artech House Publishers USA in October 2009. His research interests concentrate on the biosensors (e.g. living cell sensor, DNA sensor and protein sensor) and BioMEMS system.