Peptide Functionalized Nanoplasmonic Sensor for Explosive Detection
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In this study, a nanobiosensor for detecting explosives was developed, in which the peptide was synthesized with trinitrotoluene (TNT)-specific sequence and immobilized on nanodevice by Au–S covalent linkage, and the nanocup arrays were fabricated by nanoimprint and deposited with Au nanoparticles to generate localized surface plasmon resonance (LSPR). The device was used to monitor slight change from specific binding of 2,4,6-TNT to the peptide. With high refractive index sensing of ~104 nm/RIU, the nanocup device can detect the binding of TNT at concentration as low as 3.12 × 10−7 mg mL−1 by optical transmission spectrum modulated by LSPR. The nanosensor is also able to distinguish TNT from analogs of 2,4-dinitrotoluene and 3-nitrotoluene in the mixture with great selectivity. The peptide-based nanosensor provides novel approaches to design versatile biosensor assays by LSPR for chemical molecules.
KeywordsNanocup arrays Peptide 2,4,6-trinitrotoluene (TNT) Localized surface plasmon resonance (LSPR) Nanosensor
Explosive detections, especially for 2,4,6-trinitrotoluene (TNT), were of sustainable importance due to their threats for public security and human health as pollutants in natural water, soil, and air [1, 2, 3]. Thus, over the last couple of decades, significant efforts have been made to develop sensor devices which can detect explosive compounds rapidly, selectively, and sensitively [4, 5, 6]. These devices often used explosive sensitive materials, such as molecularly imprinted polymers, carbon nanotubes, and antibodies, to modify transducers, ranging from electrodes to fluorescent assays, for specific explosive detections. With a series of work, these devices have been demonstrated to discern explosive molecules with high sensitivity and selectivity in complex environment. Among them, biosensors attracted increasing focus because of their excellent performances in explosive detections, which might come from biological olfactory perception [7, 8, 9]. In recent studies, researches attempted to gradually integrate bio-inspired components, including whole cells, proteins, and aptamers, into various sensor platforms to memetic sniffer abilities of animals, providing a good option to replace sniffer animals in practical explosive detections.
In biosensor studies, proteins such as enzymes, antigens–antibodies, and receptors were common biosensing elements to bind target molecules and then elicit sensor responses in biochemical detections [10, 11, 12]. The proteins often had binding sites in which multiple interactions can be formed with target molecules by high specific affinities or catalysis activities. However, these complex proteins were difficult to purify and synthesize, which hindered their biosensing applications. In recent studies, peptides were widely applied as alternative biosensing materials for complex proteins in biosensor designs [13, 14, 15]. Peptides were short chains of amino acids that can be designed based on binding sites of proteins, screened into specific sequences, and finally synthesized with chemical methods. It provided an easy option to design artificial receptors to mimic molecular recognitions between proteins and analytes. The robust structures of peptides also allowed for using in more extreme environment and long-term storage, compared to natural protein molecules. Thus, peptides were ideal candidates of biosensing materials for biosensor fabrications.
However, without folding structures, peptides usually elicited slight changes in the interactions with small target molecules, which proposed high sensitivity demand for sensor methods. Recently, nanomaterials (e.g., nanoparticles, carbon nanotubes, nanoholes, and nanowires) became a new focus of ultrasensitive detection in biosensor fields [16, 17, 18, 19]. Optical, electrical, and electromechanical methods were applied to record signals from bio-functionalized nanostructures to achieve ultrasensitive detections for various chemicals. Using these nanosensors, bio-interactions could be monitored quantifiably and ultrasensitively, even at single molecular level. Among these platforms, optical detection utilizing plasmon resonance on nanostructures was one of the most common methods for nanoscale biosensors. It was also believed particularly promising because optical measurement allowed remote transduction of biomolecular binding signal without any physical connection between excitation sources and detecting elements. Thus, many biosensors were fabricated depending on plasmon resonance from nanoscale materials, such as localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering, to analyze chemicals with high sensitivities [20, 21, 22].
Here, a kind of TNT-specific peptide was designed and synthesized to modify nanocup arrays (nanoCA) for explosive detections based on LSPR. With refractive index sensing, the nanoCA could monitor binding of TNT to the peptide, while the peptide was immobilized on nanoCA by Au–S covalent linkage. In the measurement, the optical LSPR responses of the peptide-modified devices were recorded with transmission mode in the presence of explosive compounds. TNT, 2,4-Dinitrotoluene (DNT), and 3-Nitrotoluene (3-NT) were also detected at increasing concentrations, respectively. It was demonstrated that the nanosensor could detect TNT selectively in dose-dependence behavior. The mixture of TNT with DNT and 3-NT was used to show high specificity of the nanosensor using synthesized peptide. Thus, this study provided a selective and sensitive nanobiosensor platform for explosive detection.
2 Methods and Materials
2.1 Fabrication of NanoCA
2.2 Optical LSPR Measurement for NanoCA
The optical detection system was multi-mode detection platform (SpectraMax Paradigm, Molecular Devices Co., USA). Normal transmission model was applied to measure the spectra of nanoCA. As shown in Fig. 1c, the nanoCA chips were integrated into 96-well plates for high-throughput measurement. Light was emitted from the light source on the bottom of device, delivered through nanoCA chips, and received by spectrograph. During measurement, 10 μL solution containing detecting compounds was added on the surface of the chip. The small volume of the analyte formed a thin liquid layer to reduce interferences from solution itself. The scanning range was set from 300 to 900 nm and the step was fixed on 1 nm.
2.3 Synthesis and Immobilization of the Peptide
The peptide was chemically synthesized based on the reported TNT-sensitive sequence (WHWQRPLMPVSI) as bio-recognition component for TNT . An aspartic acid was added at the carboxy terminus of the chain as ‘linking residue’ tail. Then, thiol group (–SH) was added at the carboxy terminus of new sequence (WHWQRPLMPVSID) with cysteamide to generate a covalent bond of Au–S on the surface of nanoCA. The chemical synthesis of the peptide was performed with solid phase method by stepwise addition of protected amino acids to a growing peptide chain. The property of the synthesized peptide was tested by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Before the sensor experiments, the peptide was stored in the form of freeze-dried powders.
For immobilization of the peptide on the nanodevice, the peptide was dissolved in phosphate buffered saline (PBS, pH 7.4) at concentration of 250 μg mL−1. 100 μL peptide solution was spotted on the well of microplate. The solution of peptide was diffused and distributed evenly on the device due to the surface tension. After incubated for 12 h, PBS buffer was used to remove the unbonded peptide and nitrogen was used to dry the devices. Then, the nanodevice with peptide was stored under 4 °C and ready for explosive detections. All of the above immobilizing processes were performed at room temperature (22 °C).
2.4 Measurement for Explosive Detection
The standard substances of TNT, DNT, and 3-NT were purchased from Aladdin (Shanghai, China) in methanol solution at concentration of 1 mg mL−1. In explosive detections, the standard solution was diluted to five different concentrations (10−6, 10−5, 10−4, 10−3, and 10−2 mg mL−1) with methanol from the original concentration. The transmission spectrums of nanoCA were recorded by the optical system, when 30 μL analyte solution at different concentrations was added into different cavities. The all nanodevices were used once and disposable. Other reagents in experiments were analytical grades which were purchased from Aladdin (Shanghai, China).
3.1 TNT-Specific Peptide Synthesis
3.2 Optical Measurement for LSPR Property of the Nanodevice
3.3 Bio-functionalization of the NanoCA with Peptide
3.4 Explosive Measurement for Trinitrotoluene
The last decade studies about biosensors have proposed various high sensitive ways to convert bimolecular bindings into electrical and optical signals [30, 31, 32, 33]. Now, the lack of selective bio-components still remained a key challenge, making biosensors inadequate in many applications and preventing their widespread uses. Peptides provided an easy and universal approach to design bio-selectivity with artificial synthesis. However, it was difficult to detect small weight molecules by peptide-based biosensors because of slight structural changes from the specific bindings. Thus, sensor devices had to be used with complex modifications and structures to amplify binding signals. For example, quartz crystal microbalance-based sensor was often used as micro- and nanoparticles to label analyte targets for amplification of gravimetric changes [34, 35]. But, the labels might have an influence on in situ detections for certain analytes. In our study, the LSPR sensor device, nanoCA, provided an excellent platform to quantify interaction between TNT and peptide with high sensitivity, real-time response, and label-free detection. The nanostructured sensor could be combined with peptide to fabricate biosensor for TNT without complex optical coupling and any labels. The binding of TNT to peptide could be monitored selectively by LSPR generated from the periodic nanostructure in transmission measurement. In the measurement, the sensor could detect TNT at concentration as low as 3.12 × 10−7 mg mL−1. The detection limit was similar to that of several reported works using LSPR of nanomaterials for explosive detections [36, 37, 38]. It meant that the sensitivity of nanosensor combining nanocups and the peptides could satisfy explosive detections in practical applications, such as safe checking and environmental monitoring.
Besides sensitivity, the selectivity of the nanosensor was also discussed in our study. The results showed that the sensor had significant responses to TNT, low responses to DNT, and no response to 3-NT and TNP. The nanosensor showed some level of selectivity to TNT versus other chemical molecules that had similar molecular structures. It might be due to the adjacent methyl and nitro groups of molecular structure of TNT and DNT, which had electron interaction with tryptophan and histidine on peptides [39, 40]. Thus, target molecules could be connected to peptides on nanoCA and then elicit resonance wavelength shifts in detections for TNT and DNT, while 3-NT and TNP had no conjunction with the peptide and did not generate shifts in spectrum. The more groups in TNT molecules gave rise to higher affinity to peptides and more significant response signals of the sensor. Uniformly, the nanosensor also showed more significant responses to the mixture containing TNT than that without TNT in the detection for mixed explosive targets. Although the sensor had responses to DNT alone, DNT only increased the response of the sensor slightly in detection for mixture containing TNT and DNT. Those results indicated that the nanosensor could detect TNT in mixtures and would not be interfered with other compounds, even some very similar molecules.
The simple transmission measurement without complex light coupling could help to achieve low-cost and high-throughput detections for explosive compounds. As described above, the nanoCA device was fixed on the bottom of individual well of the 96-well plate, modified with TNT-specific peptides and then stored under 4 °C. Thus, 96 samples containing TNT could be analyzed simultaneously in measurement by using the microplate reader mode of multi-mode detection platform. This microplate-based measurement could not only improve the detection efficiency of the nanosensor with high-throughput characteristics, but also reduce requirement for sample volumes because of microcavities on plates. Moreover, the high-throughput measurement with microplate also provided possibility for array sensing by designing different biofunctions on the surface of nanoCA devices. Indeed, peptides have been reported by several groups to modify sensor devices for bacteria, disease biomarkers, and heavy ions [41, 42, 43]. Thus, combining with peptides designed and synthesized into different specificities, the nanobiosensor arrays could be fabricated to detect different chemical and biological molecules in a high-throughput method.
In summary, TNT-specific peptide was synthesized and used as bio-recognition components for explosive detection of TNT. NanoCA was employed to convert the binding of TNT to the peptide into LSPR responses in transmission spectrum. The nanosensor showed high sensitivity to recognize TNT at the concentration as low as 3.12 × 10−7 mg mL−1, while keeping selectivity to TNT versus DNT, 3-NT, and TNP with similar molecular structures. Even in the mixture of those three compounds, TNT could also be detected significantly. Combining the nanodevices and specifically designed peptides, this nanobiosensor platform provides a promising approach to develop versatile sensor arrays in further applications for chemical and biological analysis.
This work was supported by the National Natural Science Foundation of China (Grant No. 81371643), the Zhejiang Provincial Natural Science Foundation of China for Distinguished Young Scholars (Grant No. LR13H180002).
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