Ho Bin
Seo
a,
Young Seop
Kwon
a,
Ji-eun
Lee
a,
David
Cullen
b,
Hongseok (Moses)
Noh
*c and
Man Bock
Gu
*a
aDepartment of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-Gu, Seoul 136-713, Republic of Korea. E-mail: mbgu@korea.ac.kr
bCranfield Biotechnology Centre, Institute of BioScience and Technology, Cranfield University at Silsoe, Silsoe, Bedfordshire, UK
cDepartment of Mechanical Engineering and Mechanics, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, USA. E-mail: mosesnoh@coe.drexel.edu
First published on 14th August 2015
We present a novel reflectance-based colorimetric aptasensor using gold nanoparticles for the detection of oxytetracycline for the first time. It was found that the reflectance-based measurement at two wavelengths (650 and 520 nm) can generate more stable and sensitive signals than absorbance-based sensors to determine the aggregation of AuNPs, even at high AuNP concentrations. One of the most common antibacterial agents, oxytetracycline (OTC), was detected at concentrations as low as 1 nM in both buffer solution and tap water, which was 25-fold more sensitive, compared to the previous absorbance-based colorimetric aptasensors. This reflectance-based colorimetric aptasensor using gold nanoparticles is considered to be a better platform for portable sensing of small molecules using aptamers.
Diverse analytical methods have been investigated to realize rapid on-site detection of antibiotic residues such as OTC from small amounts of food samples. Y-channel microfluidic devices using latex microspheres, electrochemical aptasensors, and aptasensors using gold nanoparticles have been reported for the application.2–5 Some of the most promising sensors among them are colorimetric aptasensors using gold nanoparticles (AuNPs) because of their simple operation and easy detection with the naked eye.4 The previously reported colorimetric aptasensors use either AuNPs modified with partly complementary oligonucleotides for hybridizing with aptamers6,7 or unmodified AuNPs on which single stranded DNA (ssDNA) aptamers can be physically adsorbed.8 The aptasensors using unmodified AuNPs do not require any pre-treatment, and thus they are considered as a better approach for on-site sensing applications. When the target is added to the aptasensor, the target molecules will bind to the aptamers, resulting in the detachment of the adsorbed aptamers from AuNPs. The AuNPs will then aggregate, leading to the color change from red to purple (the absorbance peak shifts from 520 to 650 nm) because the localized surface plasmon resonance (LSPR) is changed.9 This absorbance peak shift can be detected by measuring the peak shift on reflectance signals.10
However, all of the previously reported colorimetric aptasensors using AuNPs are based on the indirect absorbance measurement, in which a spectrophotometer has been used.11 A typical measurement platform for colorimetric sensors using AuNPs is a spectrophotometer.11 The polychromic light that penetrates through the sample can be scanned over a specific wavelength range. A primary drawback of this method is a significant signal loss due to light scattering in the presence of AuNPs (particularly, at high AuNP concentrations). Thus, only a low range of AuNP concentrations could be used in the previous studies, resulting in a relatively high limit of detection (LOD) (25 nM).4
These limitations of absorbance-based detection can be overcome by employing reflectance configurations. Unlike the absorbance-based aptasensors in which the absorbance of AuNPs is analyzed indirectly from the spectrum transmitted, the reflectance-based measurement is a direct measurement of the reflected spectrum from the surface plasmon resonance of AuNPs (Fig. 1). In this approach, high AuNP concentrations are actually more desirable, since they can amplify the signal, potentially improving the sensitivity and LOD. In addition, since the reflectance-based measurement can be configured with flexible optical fibers, the system can be constructed on diverse platforms such as flow cells and microfluidic channels.2,12,13 However, there is also a challenge in reflectance-based measurements. The challenge arises from the fact that there are two types of reflections: specular reflection at the surface of the solution with no transmission into the solution and diffuse reflection in which the radiation penetrates into the solution and is reflected at the surface of particles with partial absorbance and scattering.14 When the measurement of diffuse reflection from the particles suspended in a solution is desired, the specular reflection from the solution or container surface must be minimized. This would require optimization of the incident angle and the sample container. So far, few studies have been reported using reflectance configurations as biosensors. A gold nanoparticle thin film modified with biotin was reported for monitoring biotin–avidin interactions and they used a fixed incident angle.10 There have been no reports using any reflectance configurations together with aptamers and AuNPs.
Fig. 1 The scheme of the reflectance-based aptasensor and the multi-well plate and the colorimetric aptasensor using AuNPs. |
This study presents, for the first time, a novel reflectance-based colorimetric aptasensor using gold nanoparticles for the detection of oxytetracycline. It was found that the reflectance-based measurement at two wavelengths (650 and 520 nm) can generate more stable and sensitive signals than the absorbance-based sensors to determine the aggregation of AuNPs, even at high AuNP concentrations. OTC was detected at concentrations as low as 1 nM in both buffer solution and tap water, which was 25-fold more sensitive, compared to the previous absorbance-based colorimetric aptasensors. Moreover, the sample does not need to be contained in the standard cuvettes and thus the sample loading platform can be miniaturized into portable and on-site diagnosis sensors. This novel reflectance-based aptasensor is believed to address the drawbacks of the absorbance-based sensors and thus has great potential to be developed into a portable sensor system for on-site diagnosis applications.
Fig. 2 Relative reflectance intensity at 520 and 650 nm light wavelengths for different incidence angles: (a) 20°, (b) 30°, (c) 40°, (d) 50°, (e) R650/R520 of AuNPs at different incidence angles. |
The ssDNA aptamers can be adsorbed on the AuNP surface by the electrostatic forces. The aptamers attached on the AuNP surface make a stable state, even at high salt concentrations. The ratio of AuNPs to aptamers is also a key parameter in this step. For finding an appropriate AuNP to aptamer ratio, we performed experiments using the ratios 1:0, 1:75, 1:100, 1:125, 1:150 and 1:200. As the aptamer concentration increased, the AuNPs became more stable (Fig. 3(c) and (d)). Therefore, the optimum ratio of AuNPs to aptamers was determined to be 1:125.
Fig. 4 Specificity of the reflectance-based colorimetric aptasensor. The final concentration of oxytetracycline, tetracycline, doxycycline, and diclofenac is 50 μM. |
Fig. 5 An overlay plot showing a dose-dependence of this reflectance-based aptasensor and the absorbance-based aptasensor using unmodified AuNPs for the detection of oxytetracycline. Filled circles indicated reflectance data while empty squares indicated absorbance data published in our previous study.17 The left vertical axis represents R650/R520 for the reflectance intensity ratio and the right vertical axis represents the normalized A650/A520 for the absorbance intensity ratio. |
In this reflectance-based method, the reflectance light from AuNPs, not scattering light, at a certain angle is measured. So, higher the AuNP concentrations, stronger the reflectance light obtained. Therefore, in this study we have used higher AuNP concentrations than that used in other methods such as absorbance-based colorimetry, in which the absorbance signal is more decreased at higher AuNP concentrations. In this novel reflectance-based aptasensor system, better results were obtained at higher AuNP concentrations even if the same aptamer sequence was used. With an AuNP concentration of 10 nM, the LOD was 1 nM, which is 25-fold smaller than the previous result obtained by the absorbance system. In addition, even though the AuNP concentration (10 nM) in this novel reflectance-based method was higher than the previous method (0.2–2 nM), the amount of sample required was decreased about 0.75 times because the experiments were conducted with only 60 μl solution.
In order to be used for on-site applications, this sensor should be functional in any sample solutions. The detection of oxytetracycline was attempted in a tap water solution for this purpose (Fig. 6). The OTC was dissolved in tap water at different concentrations. All other conditions were the same as the binding assay described in Fig. 3. Even in tap water, the LOD remained as low (1 nM) as in the buffer solution.
Fig. 6 Dose-dependent measurement of oxytetracycline using this reflectance-based aptasensor in tap water. |
Table 1 shows the comparison of the current reflectance-based aptasensor with absorbance-based aptasensors and other biosensors (cantilever sensors, light scattering agglutination assay, and indirect competitive assay) with regard to the sensor performance for the detection of OTC. LOD, the limit of quantification (LOQ), linear dynamic range, and EC50 values of the sensors were obtained from the literature.2,4,18,19 The linear range of the reflectance-based aptasensor was 0–10 nM. The reflectance-based aptasensor shows lower LOD/LOQ and does not require high sample volume or pre-treatment. Therefore, it seems to be suitable for on-site analysis of low concentration targets such as OTC. The more complicated food sample tests could be a subject of further study.
Pros | Cons | LOD/LOQ | Dynamic range | EC50 | Ref. | |
---|---|---|---|---|---|---|
Reflectance-based aptasensor | Low LOD | 1 nM/4 nM | 1 nM–1 μM | 188 nM | This study | |
Low sample vol. | ||||||
No pre-treatment | ||||||
Absorbance-based aptasensor | No pre-treatment | High LOD | 25 nM/— | 0.025–1 μM | 313 nM | 4 |
High sample vol. | ||||||
Cantilever sensor | Low LOD | Pre-treatment | 0.2 nM/— | 1–100 nM | 30 nM | 18 |
Long measuring time | ||||||
Light scattering agglutination assay | Real-time monitoring | High LOD | 217 nM/— | 0.217–21.7 μM | — | 2 |
Indirect competitive assay | High recovery rate in spiked milk | High LOD | 27 nM/ 108 nM | — | — | 19 |
Pre-treatment |
Footnote |
† Electronic supplementary information (ESI) available: Fig. S1 and S2. See DOI: 10.1039/c5an00726g |
This journal is © The Royal Society of Chemistry 2015 |