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A paper-supported aptasensor for total IgE based on luminescence resonance energy transfer from upconversion nanoparticles to carbon nanoparticles
Sensors and Actuators B 239 (2017) 319–324
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A paper-supported aptasensor for total IgE based on luminescence resonance energy transfer from upconversion nanoparticles to carbon nanoparticles Ping Jiang a , Mengyuan He b , Lin Shen b , Anni Shi b , Zhihong Liu b,c,∗ a
Department of General Surgery, Zhongnan Hoapital of Wuhan University, 169 Donghu Road, Wuhan 430072, PR China Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China c State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China b
a r t i c l e
i n f o
Article history: Received 17 May 2016 Received in revised form 10 July 2016 Accepted 1 August 2016 Available online 3 August 2016 Keywords: Paper-based sensor IgE Aptamer Upconversion nanoparticles Carbon nanoparticles
a b s t r a c t A paper-supported aptasensor was constructed for total IgE using a luminescence resonance energy transfer (LRET) protocol with upconversion nanoparticles (UCNPs) as energy donors and carbon nanoparticles (CNPs) as energy acceptors. This is the first time that zero-dimensional carbon nanoparticles were used as energy acceptors for paper-based LRET assays. The - stacking interaction between the aptamer and CNPs brought the energy donor (UCNPs) and energy acceptor (CNPs) in close proximity, induced the LRET process on the surface of paper and thus led to the luminescence quenching of UCNPs. The introduction of IgE inhibited the energy transfer and hence recovered the luminescence of UCNPs in a concentration-dependent manner, as a result of the recognition between IgE and aptamer. This aptasensor can be used to detect IgE concentration in the range of 0.5–80 ng/mL in both buffer solution and human serum samples. The IgE concentrations measured by our method were well correlated to those obtained from chemiluminescence-based clinical assay. Owing to its simplicity and accuracy, the proposed sensor thus showed the potential of clinical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Paper-based assay hold great promise to practical cost-effective applications, such as point-of-care testing and consumer diagnostics, especially in under-developed regions lacking the laboratory resources. This is particularly owing to the apparent advantages of paper substrate such as low cost, three-dimensional fibrous structures and large surface area, easy patterning and chemical modification, and the capillary wicking action for fluid flow [1–3]. Up to now, paper-based sensors have been used in medical diagnosis, environmental monitoring, and food quality control and so on [4–6]. In recent years, an increasing number of researchers have sought to integrate nanomaterials with paper-based assay formats. The rapidly developing nanotechnology can improve the quality of the paper-based devices due to the unique properties of nanomaterials. So far, AuNPs [7], magnetic nanoparticles [8], ceria
∗ Corresponding author at: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China. E-mail address: [email protected] (Z. Liu). http://dx.doi.org/10.1016/j.snb.2016.08.005 0925-4005/© 2016 Elsevier B.V. All rights reserved.
nanoparticles [9], quantum dots (QDs) [10], UCNPs [11] and carbon materials [12–16] etc. have been employed in paper-based sensors and used as labels, carriers or other functions such as surface enhanced raman scattering (SERS), surface plasmon resonance (SPR) [17,18]. UCNPs are promising luminescent materials for the construction of biosensor in body fluids owing to their features of excitation with near-infrared (NIR) light and anti-Stokes emission, which can circumvent the problem of autofluorescence and/or light scattering [19–21]. In consideration of the high complexity of both clinical samples and paper substrates, UCNPs would be particularly suitable for paper-based clinical analytics. To date, UCNPs have been successfully used as fluorescence signal reporter or energy donor in luminescence resonance energy transfer (LRET) system in some literatures [22–25]. Nonetheless, the research on paper-based analytical device based on UCNPs (UC-PADs) is just on its initial stage and needs further improvement in many aspects, such as the flexibility and diversity of assay model, the accuracy and robustness in clinical samples. On the other hand, carbon materials, such as graphite, carbon nanotubes (CNTs), carbon nanofibers (CNFs), and graphene oxide (GO) have been verified as effective energy accep-
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tors in solid assays [26]. There are also reports using GO as the energy acceptor to detect DNA, protein and pathogen on the surface of paper [27–29]. However, no report using CNPs as energy acceptor in solid matrix has been made known to public. In this work, we report a paper-supported aptamer biosensor for IgE detection using UCNPs and CNPs as the energy donor-acceptor pair. To the best of our knowledge, this is the first report using CNPs in the solid matrix as the acceptor for energy transfer. We chose immunoglobulin E (IgE), which plays a major role in allergic diseases [30], as the model analyte and the D17.4 IgE aptamer as recognition unit. This aptasensor not only reduces the background signal due to the “off-on” switching model during the sensing but also avoids the pretreatment of samples (owing to the unique merit of UCNPs), providing high sensitivity and specificity. 2. Materials and method
temperature, the resulting nanoparticles were precipitated out by the addition of equal volume of ethanol, collected by centrifugation, and washed several times with ethanol and cyclohexane (v/v, 4:1). Finally, the synthesized OA-NaYF4 : Yb, Er nanoparticles were redispersed in chloroform before further treatment. A ligand exchange strategy was used to obtain PAA-UCNPs. Briefly, 30 mL diethylene glycol (DEG) and 600 mg PAA were added to a 100 mL three-necked flask simultaneously and the mixed solution was vacuumed and heated to 110 ◦ C. 8 mL of OA-UCNPs solution was added and the mix solution reacted at 110 ◦ C for 1 h, followed by heating to 290 ◦ C and maintaining at this temperature for 6 h under argon atmosphere. After cooling down to room temperature, the resulting nanoparticles were precipitated out by the addition of equal volume of ethanol, collected by centrifugation, and washed several times with ethanol and water. The product was dried under vacuum before use.
2.1. Chemicals and reagents
2.4. Synthesis of carbon nanoparticles
Immunoglobulin E was purchased from Shanghai LincBio Science Co., Ltd. (Shanghai, China). Polyacrylic acid (PAA, with an average molecular weight of 1800), 1-(3hydrochloride dimethylaminopropyl)-3-ethylcarbodiimide (EDC·HCl), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS), 2-(N-morpholino) ethanesulfonic acid (MES), 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) and tris (hydroxymethyl) aminomethane (Tris) were from Sigma-Aldrich. Human IgG, lysozyme and bovine serum albumin (BSA) were from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). The rest of the chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and at least of analytical grade. All aqueous solutions were prepared using ultrapure water (Mill-Q, Millipore, 18.2 M resistivity). Amine modified IgE aptamer (5 -NH2 AAAAAGGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC-3 ), with five A bases next to the amine group as the spacer and the rest bases as the IgE aptamer, was supplied by Sangon Biotechnology Co., Ltd (Shanghai, China).
Carbon nanoparticles (CNPs) were synthesized with candle soot as starting material according to the reported method with some modifications [31]. Briefly, 18 mg of candle soot was added to the mixture solution containing 9 mL of HNO3 and 9 mL of N, NDimethylformamide (DMF), and then the solution was refluxed and stirred at 100 ◦ C for 18 h. After the mixture was cooled down to room temperature, a precipitate was obtained by centrifuging and washed with water for three times. Then the product was dried under vacuum and redispersed in water before use. The concentration of CNPs was calculated as 0.5 mg/mL.
2.2. Instrumentation The size and morphology of UCNPs and CNPs on the surface of paper were characterized by Zeiss SIGMA FESEM (Carl Zeiss, Germany). The crystal phase of UCNPs was identified by X’Pert Pro X-Ray Diffractometer (XRD) (PANalytical, Holland) with 2 range from 10◦ to 80◦ at a scanning rate of 4◦ per minute, with Cu K␣ irradiation (k = 1.5406 Å). UV–vis absorption spectra data were recorded with an UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan). Fluorescence spectra were collected with a RF-5301PC fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) with an external 980 nm CW laser (Beijing Hi-Tech Optoelectronic Co., Ltd.). 2.3. Synthesis of upconversion nanoparticles NaYF4 : Yb, Er upconversion nanoparticles were synthesized according to the previously reported method [20,21]. Firstly, oleic acid-stabilized UCNPs were synthesized by a solvothermal method. Rare-earth stearate Ln(oleate)3 (Y/Yb/Er = 0.80: 0.18: 0.02, moleto-mole ratio) was used as the precursor for the synthesis of oleic acid-stabilized UCNPs (OA-UCNPs). Briefly, 1 mmol of Ln(oleate)3 , 20 mmol of NaF, 10 mL of oleic acid (OA) and 10 mL of 1-octadecene (ODE) were added to a 100 mL three-necked flask simultaneously and the mixed solution reacted at 110 ◦ C with magnetic stirring for 1 h under an argon flow to obtain a transparent yellow solution, followed by heating to 290 ◦ C and maintaining at this temperature for 2 h under argon atmosphere. After cooling down to room
2.5. Attachment of the IgE aptamer to UCNPs The amino modified IgE aptamer was covalently conjugated to PAA-UCNPs using EDC·HCl and Sulfo-NHS as the cross-linking agents according to previous works [32]. Briefly, 1 mg of PAAUCNPs was dissolved in 1 mL of MES buffer solution (10 mM, pH 5.5). Then 0.6 mg of EDC·HCl and 1.2 mg of Sulfo-NHS were added to the solution and the mixture solution was incubated at room temperature with gentle shaking for 40 min to activate the carboxyl groups of PAA-UCNPs. The activated PAA-UCNPs were collected after washing with water for three times. The obtained precipitate was redispersed in 1 mL of HEPES buffer solution (10 mM, pH 7.2) containing 1 nmol of IgE aptamer. Then the mixture was maintained overnight at room temperature with gentle shaking and 10 mg of Tris was added to the mixture to block the excess NHS. The as-prepared UCNPs-IgE aptamer conjugate was harvested by centrifugation and washed with ultrapure water for three times. Finally, the product was re-dispersed in 1 mL of PB buffer solution (10 mM, pH 7.4) and stored at 4 ◦ C for further use. The concentration of UCNPs-IgE aptamer was calculated as 1 mg/mL. 2.6. Construction of paper-supported upconversion fluorescence detection devices The permanent markers were used to directly plot papersupported analytical devices with the aid of plastic templates with specific pattern [33,34]. We plotted the pattern on the whatman no. 1 paper according to template with a permanent marker and left the resultant patterned paper at room temperature to evaporate the solvent. The resins remaining in the marks in paper would form the hydrophobic barriers to separate the independent test zones. The upconversion luminescence signals were measured by self-made device. Briefly, the paper loaded with reagents and samples in the test zones was stuck on the surface of solid sample holder and placed in quartz cuvette diagonally followed by recording the
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Scheme 1. Schematic illustration of the paper-supported aptasensor for IgE detection using UCNPs and CNPs as energy donor-acceptor pair.
fluorescence intensity at a selected wavelength (546 nm for our UCNPs) with a 980 nm CW laser as the light source. 2.7. Procedure for the measurement of IgE In the luminescence quenching experiments, varying amounts of CNPs were individually added into the test zones with fixed concentration of UCNPs-IgE aptamer (0.05 mg/mL), and then the paper device was incubated for 40 min at room temperature. Subsequently, the upconversion luminescence measurements were carried out. For the determination of IgE, different concentrations of IgE were added to test zones with fixed concentration of UCNPsIgE aptamer (0.05 mg/mL) and the paper device was incubated for 30 min at 37 ◦ C. Then the CNPs were added to the test zones device with a concentration of 0.04 mg/mL followed by incubating for another 20 min. To examine the specificity of the UC-PADs aptasensor, a list of other interfering species including metal ions and proteins were added into the detection system in place of IgE following the same experimental procedures. For the determination of IgE in human serum samples, newly obtained serum from healthy people was 10-fold diluted with PBS buffer (0.01 M, 0.15 M NaCl, pH 7.4) and used as the assay medium. An identical detection procedure as in buffer was followed for IgE. 3. Results and discussions 3.1. Detection principle The UC-PADs aptasensor was constructed based on the conformation change of IgE aptamer before and after interacting with IgE (Scheme 1). The read-out signal is modulated by the high-affinity recognition of IgE with its aptamer decorated on UCNPs. In the absence of IgE, the UCNPs-aptamer loaded on the surface of paper interacts with CNPs through - stacking interaction, leading to the quenching of luminescence. While in the presence of IgE, the aptamer recognizes IgE specifically and forms aptamer/IgE complex accompanied with the conformational change. Under this circumstance, the distance between UCNPs and CNPs is enlarged, blocking the energy transfer. As a consequence, the emission of UCNPs is restored in an IgE concentration-dependent manner. 3.2. Characterization of UCNPs and CNPs NaYF4 : Yb, Er was selected as the energy donor for this aptasensor and PAA was used as surface ligand. The crystalline phase of UCNPs was characterized by X-ray diffraction (XRD) pattern (Fig. 1A) and the result shows that the peak positions and intensities agreed well with the standard values of pure hexagonalphase NaYF4 nanocrystals (JCPDS 16-0334). The existence of PAA
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molecules on the surface of UCNPs was confirmed with FT-IR spectra (Fig. 1B). As compared to bare UCNPs, the PAA-UCNPs showed unique absorption peaks of methylene asymmetric C H stretching (2969 cm−1 ), C O stretching vibration (1719 cm−1 ), carboxylate asymmetric and symmetric COO− stretching (1519 cm−1 ), OH bending vibration (1460 cm−1 ) and C O stretching (1275 cm−1 ), which demonstrated the successful attachment of PAA molecules. The size and micromorphology of as-prepared UCNPs and CNPs were characterized by scanning electron microscopic (SEM). The SEM image in Fig. 1C shows that the spherical UCNPs, which possess fairly uniform size and an average diameter of 55 nm, adhered to the surface of cellulose filter and aggregated to some extent after dropping the particles on the test zone. The SEM image in Fig. 1D also shows the spherical morphology of CNPs with the diameter ranging from 30 to 50 nm. The photo of CNPs presented in Fig. S1a shows that CNPs possessed good water-dispersibility and could keep stable for at least two weeks. The zeta potential of carbon nanoparticles was −24.0 mV (Fig. S1b), which suggests they have a negative surface charge due to the existence of oxygenated functional groups such as carboxyl and hydroxyl. As shown in Fig. S2, the emission spectrum of UCNPs overlaps well with the UV–vis absorption of the as-prepared CNPs which covers a wide band in the UV to visible region, thus facilitating the resonance energy transfer from UCNPs to CNPs. 3.3. Configuration of the paper-supported sensors The paper-supported sensors were patterned on a piece of whatman no. 1 filter with a simple plotting method. By optimizing the nib size of permanent marker, the diameter of the test zones can be reduced to 2 mm. It only required ca. 2 L of solution to fill the zone with uniformity (Fig. S3). This optimization is useful since it reduces the consumption of reagents and shortens the reaction time effectively. More importantly, full-scale UCNPs can be excited by the NIR laser beam in the small test zone, which is in favour of signal stability (Fig. S4). The paper matrix loaded with reagents and samples in the test zone was stuck on the surface of a solid sample holder and placed in quartz cuvette diagonally followed by measuring the luminescence intensity at a selected wavelength with a 980 nm CW laser as the light source (Fig. S5). To ensure the practical applicability of the sensor, we paid special attention to the reproducibility of the detecting signal. We first examined the possible position deviation of sticking the paper matrix on the sample holder. To this end, we repeatedly pasted the same test zone on the holder for seven times and detected the luminescence intensity of UCNPs in the zones, and we observed negligible alteration of the signal (RSD = 3.86%) (Fig. S6a). Also, the luminescence intensity of UCNPs (with a fixed concentration) in seven different test zones keeps almost constant (RSD = 1.03%) (Fig. S6b). 3.4. LRET between UCNPs labelled with IgE aptamer and CNPs We then investigated the quenching of UCNPs luminescence by CNPs on the surface of paper. Fig. 2A demonstrates the upconversion luminescence of UCNPs in the presence of varying amounts of CNPs. The luminescence is seen to gradually decrease with increasing concentration of CNPs. The quenching efficiency reaches to a maximum of 78.4% with 0.04 mg/mL CNPs and remains almost unchanged with further increase of the concentration of CNPs. The luminescent images of UCNPs on the surface of paper before and after quenching demonstrate that the luminescence quenching can be distinguished by naked eyes (Fig. S7). The time dependence of the luminescence quenching was investigated and is presented in Fig. 2B. The luminescence of UCNPs was quenched to the maximal degree after 15 min of reaction. As compared to a similar UCNPsCNPs LRET system in aqueous solution, which needed about 2 h to
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Fig. 1. (A) XRD pattern of PAA-UCNPs and the standard pattern of hexagonal phase NaYF4 (JCPDS 16-0334). (B) The FT-IR spectra of bare UCNPs and PAA-UCNPs. (C) SEM image of PAA-UCNPs on the surface of paper. (D) SEM image of CNPs on the surface of paper.
Fig. 2. (A) Upconversion luminescence spectra of UCNPs-IgE aptamer (0.05 mg/mL) in the presence of different concentrations of CNPs. Inset: quenching efficiency versus CNPs concentration (0, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.05 mg/mL). (B) Luminescence quenching of UCNPs-IgE aptamer (0.05 mg/mL) by 0.04 mg/mL CNPs as a function of time.
reach the maximal quenching [35], the reaction of the present sensor on the paper matrix is remarkably speeded up due to the porous structure of cellulose filter and the capillary wicking effect, which increases the local concentration of nanoparticles and accelerates the diffusion-controlled kinetics.
3.5. IgE detection in aqueous buffer The UCNPs-aptamer immobilized on the test zones of the paper were added with different concentrations of IgE and incubated for a certain time, followed by the introduction of 0.04 mg/mL CNPs. Considering that LRET is a process highly dependent on the distance between the luminophore and the quencher, the energy transfer process will be blocked once the aptamer changes its conformation upon binding with target, which impairs the - stacking interaction. As shown in Fig. 3, a gradual recovery of UCNPs luminescence was observed with increasing the concentration of IgE. Within the range of 0.5–80 ng/mL, a linear calibration with a correlation coefficient of 0.9923 was obtained between the target concentration and the relative luminescence intensity, (F−F0 )/F0 , where F and F0 were
the luminescence intensities in the presence and in the absence of IgE, respectively. To examine the specificity of the UC-PADs aptasensor, a list of other interfering species including Na+ , IgG, BSA, lysine, thrombin and lysozyme were added into the UCNPs-aptamer-CNPs aptasensor in place of IgE under the same detecting conditions. Due to the highly specific recognition between the aptamer and the target, we observed no obvious restoration of luminescence of the UCNPs (Fig. 4). Therefore, the influence of all the interfering species can be neglected, indicating the good specificity of this UC-PADs aptasensor for IgE. 3.6. Assay of IgE in human serum samples To estimate the applicability of our UC-PADs aptasensor in complicated biological matrix, we also performed IgE assay in 10-fold diluted healthy human serum. As shown in Fig. 5, a good linear calibration curve with a correlation coefficient of 0.9902 was also obtained for IgE in the range of 0.5–80 ng/mL with slightly larger standard deviations as compared to the assay in buffer solution. To evaluate the accuracy of the developed UC-PADs aptasensor, four
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Fig. 3. (A) Upconversion luminescence spectra of the test zones with various concentrations of IgE (0, 0.5, 5, 10, 20, 30, 50, 80 ng/mL). (b) Linear dependence of the relative luminescence intensity (F-F0 )/F0 on the concentration of IgE within the range of 0.5–80 ng/mL. Experiments were performed in PBS buffer (0.01 M, 0.15 M NaCl, pH 7.4). Table 1 Comparison of the detection results between clinical method and the UC-PADs aptasensor. Sample (No.)
Cclinical (ng/mL)
CUC-PADs (ng/mL)
CUC-PADs /Cclinical (%)
RSD of UC-PADs (%)
1 2 3 4
42.24 43.2 5.88 23.88
52.05 45.77 6.41 22.71
123.2 105.9 109 95.1
11.7 9.6 4.16 6.0
could be used in serum samples and may find application in clinical analysis and diagnosis. 4. Conclusions
Fig. 4. The relative intensity of test zones added with UCNPs-aptamer-CNPs plus different substances. The concentration of IgE was 80 ng/mL and that of other interfering species was 400 ng/mL. Blank represents the zone added with only UCNPs-aptamer-CNPs.
human serum samples with known IgE concentration (detected with chemiluminescence kit in the hospital) were obtained from School of Medicine, Wuhan University and were detected by our UC-PADs aptasensor with reasonable dilutions. The content of total IgE in these samples were calculated with the above calibration curve and the results are shown in Table 1. The percentage of consistency between the UC-PADs sensor and clinical method ranged from 95.1% to 123.2%, which suggests that the UC-PADs aptasensor
In summary, we have constructed a paper-supported sensor using UCNPs as the energy donors and CNPs as energy acceptors for highly sensitive and specific detection of IgE. CNPs were used as energy acceptors in LRET assay on the paper matrix for the first time. The - stacking interaction between the aptamer and CNPs induced the LRET process on the surface of paper and quenched luminescence of UCNPs. The introduction of IgE recovered the luminescence as a result of the recognition between IgE and its aptamer. A good linear calibration was obtained for IgE both in aqueous buffer and in 10-fold diluted human serum samples. The IgE concentrations measured by this sensor were well correlated to those obtained from clinical assays in hospital. The paper-supported aptasensor based on UCNPs-CNPs LRET assay may be further developed for clinical applications in the future.
Fig. 5. (A) Upconversion luminescence spectra of the test zones with various concentrations of IgE (0, 0.5, 5, 10, 20, 30, 50, 80 ng/mL). (b) Linear dependence of the relative intensity (F-F0 )/F0 on the concentration of IgE within the range of 0.5–80 ng/mL. Experiments were performed in human serum 10-fold diluted with PBS buffer (0.01 M, 0.15 M NaCl, pH 7.4).
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Acknowledgements The authors acknowledge the financial support from the National Natural Science Foundation of China (No. 21375098). 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.2016.08.005. References [1] A.W. Martinez, S.T. Phillips, G.M. Whitesides, Diagnostics for the developing world: microfluidic paper-based analytical devices, Anal. Chem. 82 (2010) 3–10. [2] X. Li, D.R. Ballerini, W. Shen, A perspective on paper-based microfluidics-current status and future trends, Biomicrofluidics 6 (2012) 011301. [3] D.M. Cate, J.A. Adkins, J. Mettakoonpitak, C.S. Henry, Recent developments in paper-based microfluidic devices, Anal. Chem. 87 (2015) 19–41. [4] C.D. Chin, V. Linder, S.K. Sia, Commercialization of microfluidic point-of-care diagnostic devices, Lab Chip 12 (2012) 2118–2134. [5] A.W. Martinez, S.T. Phillips, E. Carrilho, S.W. Thomas, H. Sindi, G.M. Whitesides, Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time off-site diagnosis, Anal. Chem. 80 (2008) 3699–3707. [6] X. Chen, J. Chen, F.B. Wang, X. Xiang, M. Luo, X.H. Ji, Z.K. He, Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices, Biosens. Bioelectron. 35 (2012) 363–368. [7] W. Zhao, M.M. Ali, S.D. Aguirre, M.A. Brook, Y. Li, Paper-based bioassays using gold nanoparticle colorimetric probes, Anal. Chem. 80 (2008) 8431–8437. [8] S. Puertas, M. Moros, R. Fernández-Pacheco, M.R. Ibarra, V. Grazú, J.M. de la Fuente, Designing novel nano-immunoassays: antibody orientation versus sensitivity, J. Phys. D: Appl. Phys. 43 (2010) 474012. [9] M. Ornatska, E. Sharpe, D. Andreescu, S. Andreescu, Paper bioassay based on ceria nanoparticles as colorimetric probes, Anal. Chem. 83 (2011) 4273–4280. [10] X. Zhu, L. Chen, P. Shen, J. Jia, D. Zhang, L. Yang, High sensitive detection of Cry1Ab protein using a quantum dot-based fluorescence-linked immunosorbent assay, J. Agric. Food Chem. 59 (2011) 2184–2189. [11] F. Zhou, M.O. Noor, U.J. Krull, Luminescence resonance energy transfer-based nucleic acid hybridization assay on cellulose paper with upconverting phosphor as donors, Anal. Chem. 86 (2014) 2719–2726. ˚ [12] M. Blaˇzková, B. Javurková, L. Fukal, P. Rauch, Immunochromatographic strip test for detection of genus Cronobacter, Biosens. Bioelectron. 26 (2011) 2828–2834. [13] D.P. Kalogianni, L.M. Boutsika, P.G. Kouremenou, T.K. Christopoulos, P.C. Ioannou, Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization, Anal. Bioanal. Chem. 400 (2011) 1145–1152. [14] Q.S. Mei, Z.P. Zhang, Photoluminescent graphene oxide ink to print sensors onto microporous membranes for versatile visualization bioassays, Angew. Chem. Int. Ed. 51 (2012) 5602–5606. [15] B. Kim, Y.J. Lu, T. Kim, J.W. Han, M. Meyyappan, J. Li, Carbon nanotube coated paper sensor for damage diagnosis, ACS Nano 8 (2014) 12092–12097. [16] Y.J. Kim, G. Jang, T.S. Lee, New fluorescent metal-ion detection using a paper-based sensor strip containing tethered rhodamine carbon nanodots, ACS Appl. Mater. Interfaces 7 (2015) 15649–15657. [17] C. Parolo, A. Merkoc¸i, Paper-based nanobiosensors for diagnostics, Chem. Soc. Rev. 42 (2013) 450–457. [18] S. Song, Y. Qin, Y. He, Q. Huang, C. Fan, H.Y. Chen, Functional nanoprobes for ultrasensitive detection of biomolecules, Chem. Soc. Rev. 39 (2010) 4234–4243. [19] Y. Xiao, L.Y. Zeng, Z.J. Wu, Z.H. Liu, Construction of an upconversion nanoprobe with few-atom silver nanoclusters as the energy acceptor, Angew. Chem. Int. Ed. 54 (2015) 5323–5327. [20] Z. Li, S.W. Lv, Y.L. Wang, S.Y. Chen, Z.H. Liu, Construction of LRET-based nanoprobe using upconversion nanoparticles with confined emitters and bared surface as luminophore, J. Am. Chem. Soc. 137 (2015) 3421–3427.
[21] Z. Li, T. Liang, S.W. Lv, Q.G. Zhuang, Z.H. Liu, A rationally designed upconversion nanoprobe for in vivo detection of hydroxyl radical, J. Am. Chem. Soc. 137 (2015) 11179–11185. [22] Q. Ju, U. Uddayasankar, U.J. Krull, Paper-based DNA detection using lanthanide-doped LiYF4 upconversion nanocrystals as bioprobe, Small 10 (2014) 3912–3917. [23] Q.S. Mei, H.R. Jing, Y. Li, W. Yisibashaer, J. Chen, B.N. Li, Y. Zhang, Smartphone based visual and quantitative assays on upconversional paper sensor, Biosens. Bioelectron. 75 (2016) 427–432. [24] S. Doughan, U. Uddayasankar, U.J. Krull, A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors, Anal. Chim. Acta 878 (2015) 1–8. [25] M.Y. He, Z. Li, Y.Y. Ge, Z.H. Liu, Portable upconversion nanoparticles-based paper device for field testing of drug abuse, Anal. Chem. 88 (2016) 1530–1534. [26] E. Morales-Narváez, B. Pérez-López, L.B. Pires, A. Merkoc¸i, Simple Förster resonance energy transfer evidence for the ultrahigh quantum dot quenching efficiency by graphene oxide compared to other carbon structures, Carbon 50 (2010) 2987–2993. [27] E. Morales-Narváez, A. Hassan, A. Merkoc¸i, Graphene oxide as a pathogen-revealing agent: sensing with a digital-like response, Angew. Chem. Int. Ed. 52 (2013) 13779–13783. [28] E. Morales-Narváez, T. Naghdi, E. Zor, A. Merkoc¸i, Photoluminescent lateral-flow immunoassay revealed by graphene oxide: highly sensitive paper-based pathogen detection, Anal. Chem. 87 (2015) 8573–8577. [29] H. Li, X.E. Fang, H.M. Cao, J.L. Kong, Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins, Biosens. Bioelectron. 80 (2016) 79–83. [30] B.Y. Jiang, F.Z. Li, C.Y. Yang, J.Q. Xie, Y. Xiang, R. Yuan, Aptamer pseudoknot-functionalized electronic sensor for reagentless and single-step detection of immunoglobulin E in human serum, Anal. Chem. 87 (2015) 3094–3098. [31] H.L. Li, Y.W. Zhang, L. Wang, J.Q. Tian, X.P. Sun, Nucleic acid detection using carbon nanoparticles as a fluorescent sensing platform, Chem. Commun. 47 (2011) 961–963. [32] Y.H. Wang, L. Bao, Z.H. Liu, D.W. Pang, Aptamer biosensor based on fluorescence resonance energy transfer from upconverting phosphors to carbon nanoparticles for thrombin detection in human plasma, Anal. Chem. 83 (2011) 8130–8137. [33] J.F. Nie, Y. Zhang, L.W. Lin, C.B. Zhou, S.H. Li, L.M. Zhang, J.P. Li, Low-cost fabrication of paper-based microfluidic devices by one-step plotting, Anal. Chem. 84 (2012) 6331–6335. [34] M.Y. He, Z.H. Liu, Paper-based microfluidic device with upconversion fluorescence assay, Anal. Chem. 85 (2013) 11691–11694. [35] Y.H. Wang, P. Shen, C.Y. Li, Y.Y. Wang, Z.H. Liu, Upconversion fluorescence resonance energy transfer based biosensor for ultrasensitive detection of matrix metalloproteinase-2 in blood, Anal. Chem. 84 (2012) 1466–1473.
Biographies Ping Jiang received his M.S. degree from medical college of wuhan university in 2009, and has worked in general surgery department of Zhongnan hospital of Wuhan university since then. His work covers the clinical testing of blood samples and surgical operations. Mengyuan He received her B.S. degree from Civil Aviation University of China in 2011. Then she joined Prof. Zhihong Liu’s group for graduate study. Her main research interest is in the construction of paper-based analytical devices and diagnostics. Lin Shen is currently pursuing her Master’s degree at Wuhan University. Her research interest focuses on preparation and characterization of upconversion nanoparticles. Anni Shi is an undergraduate in Wuhan University. She joined Prof. Zhihong Liu’s group in 2014. Her research interest is mainly the preparation of paper-based microfluidic analytical devices. Zhihong Liu obtained Ph. D. degree from the Department of Chemistry, Wuhan University before carrying out postdoctoral research at College of Life Science, Wuhan University and Ohio State University, USA. He is currently a professor at the Department of Chemistry, Wuhan University.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A paper-supported aptasensor for total IgE based on luminescence resonance energy transfer from upconversion nanoparticles to carbon nanoparticles Ping Jiang a , Mengyuan He b , Lin Shen b , Anni Shi b , Zhihong Liu b,c,∗ a
Department of General Surgery, Zhongnan Hoapital of Wuhan University, 169 Donghu Road, Wuhan 430072, PR China Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China c State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China b
a r t i c l e
i n f o
Article history: Received 17 May 2016 Received in revised form 10 July 2016 Accepted 1 August 2016 Available online 3 August 2016 Keywords: Paper-based sensor IgE Aptamer Upconversion nanoparticles Carbon nanoparticles
a b s t r a c t A paper-supported aptasensor was constructed for total IgE using a luminescence resonance energy transfer (LRET) protocol with upconversion nanoparticles (UCNPs) as energy donors and carbon nanoparticles (CNPs) as energy acceptors. This is the first time that zero-dimensional carbon nanoparticles were used as energy acceptors for paper-based LRET assays. The - stacking interaction between the aptamer and CNPs brought the energy donor (UCNPs) and energy acceptor (CNPs) in close proximity, induced the LRET process on the surface of paper and thus led to the luminescence quenching of UCNPs. The introduction of IgE inhibited the energy transfer and hence recovered the luminescence of UCNPs in a concentration-dependent manner, as a result of the recognition between IgE and aptamer. This aptasensor can be used to detect IgE concentration in the range of 0.5–80 ng/mL in both buffer solution and human serum samples. The IgE concentrations measured by our method were well correlated to those obtained from chemiluminescence-based clinical assay. Owing to its simplicity and accuracy, the proposed sensor thus showed the potential of clinical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Paper-based assay hold great promise to practical cost-effective applications, such as point-of-care testing and consumer diagnostics, especially in under-developed regions lacking the laboratory resources. This is particularly owing to the apparent advantages of paper substrate such as low cost, three-dimensional fibrous structures and large surface area, easy patterning and chemical modification, and the capillary wicking action for fluid flow [1–3]. Up to now, paper-based sensors have been used in medical diagnosis, environmental monitoring, and food quality control and so on [4–6]. In recent years, an increasing number of researchers have sought to integrate nanomaterials with paper-based assay formats. The rapidly developing nanotechnology can improve the quality of the paper-based devices due to the unique properties of nanomaterials. So far, AuNPs [7], magnetic nanoparticles [8], ceria
∗ Corresponding author at: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China. E-mail address: [email protected] (Z. Liu). http://dx.doi.org/10.1016/j.snb.2016.08.005 0925-4005/© 2016 Elsevier B.V. All rights reserved.
nanoparticles [9], quantum dots (QDs) [10], UCNPs [11] and carbon materials [12–16] etc. have been employed in paper-based sensors and used as labels, carriers or other functions such as surface enhanced raman scattering (SERS), surface plasmon resonance (SPR) [17,18]. UCNPs are promising luminescent materials for the construction of biosensor in body fluids owing to their features of excitation with near-infrared (NIR) light and anti-Stokes emission, which can circumvent the problem of autofluorescence and/or light scattering [19–21]. In consideration of the high complexity of both clinical samples and paper substrates, UCNPs would be particularly suitable for paper-based clinical analytics. To date, UCNPs have been successfully used as fluorescence signal reporter or energy donor in luminescence resonance energy transfer (LRET) system in some literatures [22–25]. Nonetheless, the research on paper-based analytical device based on UCNPs (UC-PADs) is just on its initial stage and needs further improvement in many aspects, such as the flexibility and diversity of assay model, the accuracy and robustness in clinical samples. On the other hand, carbon materials, such as graphite, carbon nanotubes (CNTs), carbon nanofibers (CNFs), and graphene oxide (GO) have been verified as effective energy accep-
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tors in solid assays [26]. There are also reports using GO as the energy acceptor to detect DNA, protein and pathogen on the surface of paper [27–29]. However, no report using CNPs as energy acceptor in solid matrix has been made known to public. In this work, we report a paper-supported aptamer biosensor for IgE detection using UCNPs and CNPs as the energy donor-acceptor pair. To the best of our knowledge, this is the first report using CNPs in the solid matrix as the acceptor for energy transfer. We chose immunoglobulin E (IgE), which plays a major role in allergic diseases [30], as the model analyte and the D17.4 IgE aptamer as recognition unit. This aptasensor not only reduces the background signal due to the “off-on” switching model during the sensing but also avoids the pretreatment of samples (owing to the unique merit of UCNPs), providing high sensitivity and specificity. 2. Materials and method
temperature, the resulting nanoparticles were precipitated out by the addition of equal volume of ethanol, collected by centrifugation, and washed several times with ethanol and cyclohexane (v/v, 4:1). Finally, the synthesized OA-NaYF4 : Yb, Er nanoparticles were redispersed in chloroform before further treatment. A ligand exchange strategy was used to obtain PAA-UCNPs. Briefly, 30 mL diethylene glycol (DEG) and 600 mg PAA were added to a 100 mL three-necked flask simultaneously and the mixed solution was vacuumed and heated to 110 ◦ C. 8 mL of OA-UCNPs solution was added and the mix solution reacted at 110 ◦ C for 1 h, followed by heating to 290 ◦ C and maintaining at this temperature for 6 h under argon atmosphere. After cooling down to room temperature, the resulting nanoparticles were precipitated out by the addition of equal volume of ethanol, collected by centrifugation, and washed several times with ethanol and water. The product was dried under vacuum before use.
2.1. Chemicals and reagents
2.4. Synthesis of carbon nanoparticles
Immunoglobulin E was purchased from Shanghai LincBio Science Co., Ltd. (Shanghai, China). Polyacrylic acid (PAA, with an average molecular weight of 1800), 1-(3hydrochloride dimethylaminopropyl)-3-ethylcarbodiimide (EDC·HCl), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS), 2-(N-morpholino) ethanesulfonic acid (MES), 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) and tris (hydroxymethyl) aminomethane (Tris) were from Sigma-Aldrich. Human IgG, lysozyme and bovine serum albumin (BSA) were from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). The rest of the chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and at least of analytical grade. All aqueous solutions were prepared using ultrapure water (Mill-Q, Millipore, 18.2 M resistivity). Amine modified IgE aptamer (5 -NH2 AAAAAGGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC-3 ), with five A bases next to the amine group as the spacer and the rest bases as the IgE aptamer, was supplied by Sangon Biotechnology Co., Ltd (Shanghai, China).
Carbon nanoparticles (CNPs) were synthesized with candle soot as starting material according to the reported method with some modifications [31]. Briefly, 18 mg of candle soot was added to the mixture solution containing 9 mL of HNO3 and 9 mL of N, NDimethylformamide (DMF), and then the solution was refluxed and stirred at 100 ◦ C for 18 h. After the mixture was cooled down to room temperature, a precipitate was obtained by centrifuging and washed with water for three times. Then the product was dried under vacuum and redispersed in water before use. The concentration of CNPs was calculated as 0.5 mg/mL.
2.2. Instrumentation The size and morphology of UCNPs and CNPs on the surface of paper were characterized by Zeiss SIGMA FESEM (Carl Zeiss, Germany). The crystal phase of UCNPs was identified by X’Pert Pro X-Ray Diffractometer (XRD) (PANalytical, Holland) with 2 range from 10◦ to 80◦ at a scanning rate of 4◦ per minute, with Cu K␣ irradiation (k = 1.5406 Å). UV–vis absorption spectra data were recorded with an UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan). Fluorescence spectra were collected with a RF-5301PC fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) with an external 980 nm CW laser (Beijing Hi-Tech Optoelectronic Co., Ltd.). 2.3. Synthesis of upconversion nanoparticles NaYF4 : Yb, Er upconversion nanoparticles were synthesized according to the previously reported method [20,21]. Firstly, oleic acid-stabilized UCNPs were synthesized by a solvothermal method. Rare-earth stearate Ln(oleate)3 (Y/Yb/Er = 0.80: 0.18: 0.02, moleto-mole ratio) was used as the precursor for the synthesis of oleic acid-stabilized UCNPs (OA-UCNPs). Briefly, 1 mmol of Ln(oleate)3 , 20 mmol of NaF, 10 mL of oleic acid (OA) and 10 mL of 1-octadecene (ODE) were added to a 100 mL three-necked flask simultaneously and the mixed solution reacted at 110 ◦ C with magnetic stirring for 1 h under an argon flow to obtain a transparent yellow solution, followed by heating to 290 ◦ C and maintaining at this temperature for 2 h under argon atmosphere. After cooling down to room
2.5. Attachment of the IgE aptamer to UCNPs The amino modified IgE aptamer was covalently conjugated to PAA-UCNPs using EDC·HCl and Sulfo-NHS as the cross-linking agents according to previous works [32]. Briefly, 1 mg of PAAUCNPs was dissolved in 1 mL of MES buffer solution (10 mM, pH 5.5). Then 0.6 mg of EDC·HCl and 1.2 mg of Sulfo-NHS were added to the solution and the mixture solution was incubated at room temperature with gentle shaking for 40 min to activate the carboxyl groups of PAA-UCNPs. The activated PAA-UCNPs were collected after washing with water for three times. The obtained precipitate was redispersed in 1 mL of HEPES buffer solution (10 mM, pH 7.2) containing 1 nmol of IgE aptamer. Then the mixture was maintained overnight at room temperature with gentle shaking and 10 mg of Tris was added to the mixture to block the excess NHS. The as-prepared UCNPs-IgE aptamer conjugate was harvested by centrifugation and washed with ultrapure water for three times. Finally, the product was re-dispersed in 1 mL of PB buffer solution (10 mM, pH 7.4) and stored at 4 ◦ C for further use. The concentration of UCNPs-IgE aptamer was calculated as 1 mg/mL. 2.6. Construction of paper-supported upconversion fluorescence detection devices The permanent markers were used to directly plot papersupported analytical devices with the aid of plastic templates with specific pattern [33,34]. We plotted the pattern on the whatman no. 1 paper according to template with a permanent marker and left the resultant patterned paper at room temperature to evaporate the solvent. The resins remaining in the marks in paper would form the hydrophobic barriers to separate the independent test zones. The upconversion luminescence signals were measured by self-made device. Briefly, the paper loaded with reagents and samples in the test zones was stuck on the surface of solid sample holder and placed in quartz cuvette diagonally followed by recording the
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Scheme 1. Schematic illustration of the paper-supported aptasensor for IgE detection using UCNPs and CNPs as energy donor-acceptor pair.
fluorescence intensity at a selected wavelength (546 nm for our UCNPs) with a 980 nm CW laser as the light source. 2.7. Procedure for the measurement of IgE In the luminescence quenching experiments, varying amounts of CNPs were individually added into the test zones with fixed concentration of UCNPs-IgE aptamer (0.05 mg/mL), and then the paper device was incubated for 40 min at room temperature. Subsequently, the upconversion luminescence measurements were carried out. For the determination of IgE, different concentrations of IgE were added to test zones with fixed concentration of UCNPsIgE aptamer (0.05 mg/mL) and the paper device was incubated for 30 min at 37 ◦ C. Then the CNPs were added to the test zones device with a concentration of 0.04 mg/mL followed by incubating for another 20 min. To examine the specificity of the UC-PADs aptasensor, a list of other interfering species including metal ions and proteins were added into the detection system in place of IgE following the same experimental procedures. For the determination of IgE in human serum samples, newly obtained serum from healthy people was 10-fold diluted with PBS buffer (0.01 M, 0.15 M NaCl, pH 7.4) and used as the assay medium. An identical detection procedure as in buffer was followed for IgE. 3. Results and discussions 3.1. Detection principle The UC-PADs aptasensor was constructed based on the conformation change of IgE aptamer before and after interacting with IgE (Scheme 1). The read-out signal is modulated by the high-affinity recognition of IgE with its aptamer decorated on UCNPs. In the absence of IgE, the UCNPs-aptamer loaded on the surface of paper interacts with CNPs through - stacking interaction, leading to the quenching of luminescence. While in the presence of IgE, the aptamer recognizes IgE specifically and forms aptamer/IgE complex accompanied with the conformational change. Under this circumstance, the distance between UCNPs and CNPs is enlarged, blocking the energy transfer. As a consequence, the emission of UCNPs is restored in an IgE concentration-dependent manner. 3.2. Characterization of UCNPs and CNPs NaYF4 : Yb, Er was selected as the energy donor for this aptasensor and PAA was used as surface ligand. The crystalline phase of UCNPs was characterized by X-ray diffraction (XRD) pattern (Fig. 1A) and the result shows that the peak positions and intensities agreed well with the standard values of pure hexagonalphase NaYF4 nanocrystals (JCPDS 16-0334). The existence of PAA
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molecules on the surface of UCNPs was confirmed with FT-IR spectra (Fig. 1B). As compared to bare UCNPs, the PAA-UCNPs showed unique absorption peaks of methylene asymmetric C H stretching (2969 cm−1 ), C O stretching vibration (1719 cm−1 ), carboxylate asymmetric and symmetric COO− stretching (1519 cm−1 ), OH bending vibration (1460 cm−1 ) and C O stretching (1275 cm−1 ), which demonstrated the successful attachment of PAA molecules. The size and micromorphology of as-prepared UCNPs and CNPs were characterized by scanning electron microscopic (SEM). The SEM image in Fig. 1C shows that the spherical UCNPs, which possess fairly uniform size and an average diameter of 55 nm, adhered to the surface of cellulose filter and aggregated to some extent after dropping the particles on the test zone. The SEM image in Fig. 1D also shows the spherical morphology of CNPs with the diameter ranging from 30 to 50 nm. The photo of CNPs presented in Fig. S1a shows that CNPs possessed good water-dispersibility and could keep stable for at least two weeks. The zeta potential of carbon nanoparticles was −24.0 mV (Fig. S1b), which suggests they have a negative surface charge due to the existence of oxygenated functional groups such as carboxyl and hydroxyl. As shown in Fig. S2, the emission spectrum of UCNPs overlaps well with the UV–vis absorption of the as-prepared CNPs which covers a wide band in the UV to visible region, thus facilitating the resonance energy transfer from UCNPs to CNPs. 3.3. Configuration of the paper-supported sensors The paper-supported sensors were patterned on a piece of whatman no. 1 filter with a simple plotting method. By optimizing the nib size of permanent marker, the diameter of the test zones can be reduced to 2 mm. It only required ca. 2 L of solution to fill the zone with uniformity (Fig. S3). This optimization is useful since it reduces the consumption of reagents and shortens the reaction time effectively. More importantly, full-scale UCNPs can be excited by the NIR laser beam in the small test zone, which is in favour of signal stability (Fig. S4). The paper matrix loaded with reagents and samples in the test zone was stuck on the surface of a solid sample holder and placed in quartz cuvette diagonally followed by measuring the luminescence intensity at a selected wavelength with a 980 nm CW laser as the light source (Fig. S5). To ensure the practical applicability of the sensor, we paid special attention to the reproducibility of the detecting signal. We first examined the possible position deviation of sticking the paper matrix on the sample holder. To this end, we repeatedly pasted the same test zone on the holder for seven times and detected the luminescence intensity of UCNPs in the zones, and we observed negligible alteration of the signal (RSD = 3.86%) (Fig. S6a). Also, the luminescence intensity of UCNPs (with a fixed concentration) in seven different test zones keeps almost constant (RSD = 1.03%) (Fig. S6b). 3.4. LRET between UCNPs labelled with IgE aptamer and CNPs We then investigated the quenching of UCNPs luminescence by CNPs on the surface of paper. Fig. 2A demonstrates the upconversion luminescence of UCNPs in the presence of varying amounts of CNPs. The luminescence is seen to gradually decrease with increasing concentration of CNPs. The quenching efficiency reaches to a maximum of 78.4% with 0.04 mg/mL CNPs and remains almost unchanged with further increase of the concentration of CNPs. The luminescent images of UCNPs on the surface of paper before and after quenching demonstrate that the luminescence quenching can be distinguished by naked eyes (Fig. S7). The time dependence of the luminescence quenching was investigated and is presented in Fig. 2B. The luminescence of UCNPs was quenched to the maximal degree after 15 min of reaction. As compared to a similar UCNPsCNPs LRET system in aqueous solution, which needed about 2 h to
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Fig. 1. (A) XRD pattern of PAA-UCNPs and the standard pattern of hexagonal phase NaYF4 (JCPDS 16-0334). (B) The FT-IR spectra of bare UCNPs and PAA-UCNPs. (C) SEM image of PAA-UCNPs on the surface of paper. (D) SEM image of CNPs on the surface of paper.
Fig. 2. (A) Upconversion luminescence spectra of UCNPs-IgE aptamer (0.05 mg/mL) in the presence of different concentrations of CNPs. Inset: quenching efficiency versus CNPs concentration (0, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.05 mg/mL). (B) Luminescence quenching of UCNPs-IgE aptamer (0.05 mg/mL) by 0.04 mg/mL CNPs as a function of time.
reach the maximal quenching [35], the reaction of the present sensor on the paper matrix is remarkably speeded up due to the porous structure of cellulose filter and the capillary wicking effect, which increases the local concentration of nanoparticles and accelerates the diffusion-controlled kinetics.
3.5. IgE detection in aqueous buffer The UCNPs-aptamer immobilized on the test zones of the paper were added with different concentrations of IgE and incubated for a certain time, followed by the introduction of 0.04 mg/mL CNPs. Considering that LRET is a process highly dependent on the distance between the luminophore and the quencher, the energy transfer process will be blocked once the aptamer changes its conformation upon binding with target, which impairs the - stacking interaction. As shown in Fig. 3, a gradual recovery of UCNPs luminescence was observed with increasing the concentration of IgE. Within the range of 0.5–80 ng/mL, a linear calibration with a correlation coefficient of 0.9923 was obtained between the target concentration and the relative luminescence intensity, (F−F0 )/F0 , where F and F0 were
the luminescence intensities in the presence and in the absence of IgE, respectively. To examine the specificity of the UC-PADs aptasensor, a list of other interfering species including Na+ , IgG, BSA, lysine, thrombin and lysozyme were added into the UCNPs-aptamer-CNPs aptasensor in place of IgE under the same detecting conditions. Due to the highly specific recognition between the aptamer and the target, we observed no obvious restoration of luminescence of the UCNPs (Fig. 4). Therefore, the influence of all the interfering species can be neglected, indicating the good specificity of this UC-PADs aptasensor for IgE. 3.6. Assay of IgE in human serum samples To estimate the applicability of our UC-PADs aptasensor in complicated biological matrix, we also performed IgE assay in 10-fold diluted healthy human serum. As shown in Fig. 5, a good linear calibration curve with a correlation coefficient of 0.9902 was also obtained for IgE in the range of 0.5–80 ng/mL with slightly larger standard deviations as compared to the assay in buffer solution. To evaluate the accuracy of the developed UC-PADs aptasensor, four
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Fig. 3. (A) Upconversion luminescence spectra of the test zones with various concentrations of IgE (0, 0.5, 5, 10, 20, 30, 50, 80 ng/mL). (b) Linear dependence of the relative luminescence intensity (F-F0 )/F0 on the concentration of IgE within the range of 0.5–80 ng/mL. Experiments were performed in PBS buffer (0.01 M, 0.15 M NaCl, pH 7.4). Table 1 Comparison of the detection results between clinical method and the UC-PADs aptasensor. Sample (No.)
Cclinical (ng/mL)
CUC-PADs (ng/mL)
CUC-PADs /Cclinical (%)
RSD of UC-PADs (%)
1 2 3 4
42.24 43.2 5.88 23.88
52.05 45.77 6.41 22.71
123.2 105.9 109 95.1
11.7 9.6 4.16 6.0
could be used in serum samples and may find application in clinical analysis and diagnosis. 4. Conclusions
Fig. 4. The relative intensity of test zones added with UCNPs-aptamer-CNPs plus different substances. The concentration of IgE was 80 ng/mL and that of other interfering species was 400 ng/mL. Blank represents the zone added with only UCNPs-aptamer-CNPs.
human serum samples with known IgE concentration (detected with chemiluminescence kit in the hospital) were obtained from School of Medicine, Wuhan University and were detected by our UC-PADs aptasensor with reasonable dilutions. The content of total IgE in these samples were calculated with the above calibration curve and the results are shown in Table 1. The percentage of consistency between the UC-PADs sensor and clinical method ranged from 95.1% to 123.2%, which suggests that the UC-PADs aptasensor
In summary, we have constructed a paper-supported sensor using UCNPs as the energy donors and CNPs as energy acceptors for highly sensitive and specific detection of IgE. CNPs were used as energy acceptors in LRET assay on the paper matrix for the first time. The - stacking interaction between the aptamer and CNPs induced the LRET process on the surface of paper and quenched luminescence of UCNPs. The introduction of IgE recovered the luminescence as a result of the recognition between IgE and its aptamer. A good linear calibration was obtained for IgE both in aqueous buffer and in 10-fold diluted human serum samples. The IgE concentrations measured by this sensor were well correlated to those obtained from clinical assays in hospital. The paper-supported aptasensor based on UCNPs-CNPs LRET assay may be further developed for clinical applications in the future.
Fig. 5. (A) Upconversion luminescence spectra of the test zones with various concentrations of IgE (0, 0.5, 5, 10, 20, 30, 50, 80 ng/mL). (b) Linear dependence of the relative intensity (F-F0 )/F0 on the concentration of IgE within the range of 0.5–80 ng/mL. Experiments were performed in human serum 10-fold diluted with PBS buffer (0.01 M, 0.15 M NaCl, pH 7.4).
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Acknowledgements The authors acknowledge the financial support from the National Natural Science Foundation of China (No. 21375098). 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.2016.08.005. References [1] A.W. Martinez, S.T. Phillips, G.M. Whitesides, Diagnostics for the developing world: microfluidic paper-based analytical devices, Anal. Chem. 82 (2010) 3–10. [2] X. Li, D.R. Ballerini, W. Shen, A perspective on paper-based microfluidics-current status and future trends, Biomicrofluidics 6 (2012) 011301. [3] D.M. Cate, J.A. Adkins, J. Mettakoonpitak, C.S. Henry, Recent developments in paper-based microfluidic devices, Anal. Chem. 87 (2015) 19–41. [4] C.D. Chin, V. Linder, S.K. Sia, Commercialization of microfluidic point-of-care diagnostic devices, Lab Chip 12 (2012) 2118–2134. [5] A.W. Martinez, S.T. Phillips, E. Carrilho, S.W. Thomas, H. Sindi, G.M. Whitesides, Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time off-site diagnosis, Anal. Chem. 80 (2008) 3699–3707. [6] X. Chen, J. Chen, F.B. Wang, X. Xiang, M. Luo, X.H. Ji, Z.K. He, Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices, Biosens. Bioelectron. 35 (2012) 363–368. [7] W. Zhao, M.M. Ali, S.D. Aguirre, M.A. Brook, Y. Li, Paper-based bioassays using gold nanoparticle colorimetric probes, Anal. Chem. 80 (2008) 8431–8437. [8] S. Puertas, M. Moros, R. Fernández-Pacheco, M.R. Ibarra, V. Grazú, J.M. de la Fuente, Designing novel nano-immunoassays: antibody orientation versus sensitivity, J. Phys. D: Appl. Phys. 43 (2010) 474012. [9] M. Ornatska, E. Sharpe, D. Andreescu, S. Andreescu, Paper bioassay based on ceria nanoparticles as colorimetric probes, Anal. Chem. 83 (2011) 4273–4280. [10] X. Zhu, L. Chen, P. Shen, J. Jia, D. Zhang, L. Yang, High sensitive detection of Cry1Ab protein using a quantum dot-based fluorescence-linked immunosorbent assay, J. Agric. Food Chem. 59 (2011) 2184–2189. [11] F. Zhou, M.O. Noor, U.J. Krull, Luminescence resonance energy transfer-based nucleic acid hybridization assay on cellulose paper with upconverting phosphor as donors, Anal. Chem. 86 (2014) 2719–2726. ˚ [12] M. Blaˇzková, B. Javurková, L. Fukal, P. Rauch, Immunochromatographic strip test for detection of genus Cronobacter, Biosens. Bioelectron. 26 (2011) 2828–2834. [13] D.P. Kalogianni, L.M. Boutsika, P.G. Kouremenou, T.K. Christopoulos, P.C. Ioannou, Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization, Anal. Bioanal. Chem. 400 (2011) 1145–1152. [14] Q.S. Mei, Z.P. Zhang, Photoluminescent graphene oxide ink to print sensors onto microporous membranes for versatile visualization bioassays, Angew. Chem. Int. Ed. 51 (2012) 5602–5606. [15] B. Kim, Y.J. Lu, T. Kim, J.W. Han, M. Meyyappan, J. Li, Carbon nanotube coated paper sensor for damage diagnosis, ACS Nano 8 (2014) 12092–12097. [16] Y.J. Kim, G. Jang, T.S. Lee, New fluorescent metal-ion detection using a paper-based sensor strip containing tethered rhodamine carbon nanodots, ACS Appl. Mater. Interfaces 7 (2015) 15649–15657. [17] C. Parolo, A. Merkoc¸i, Paper-based nanobiosensors for diagnostics, Chem. Soc. Rev. 42 (2013) 450–457. [18] S. Song, Y. Qin, Y. He, Q. Huang, C. Fan, H.Y. Chen, Functional nanoprobes for ultrasensitive detection of biomolecules, Chem. Soc. Rev. 39 (2010) 4234–4243. [19] Y. Xiao, L.Y. Zeng, Z.J. Wu, Z.H. Liu, Construction of an upconversion nanoprobe with few-atom silver nanoclusters as the energy acceptor, Angew. Chem. Int. Ed. 54 (2015) 5323–5327. [20] Z. Li, S.W. Lv, Y.L. Wang, S.Y. Chen, Z.H. Liu, Construction of LRET-based nanoprobe using upconversion nanoparticles with confined emitters and bared surface as luminophore, J. Am. Chem. Soc. 137 (2015) 3421–3427.
[21] Z. Li, T. Liang, S.W. Lv, Q.G. Zhuang, Z.H. Liu, A rationally designed upconversion nanoprobe for in vivo detection of hydroxyl radical, J. Am. Chem. Soc. 137 (2015) 11179–11185. [22] Q. Ju, U. Uddayasankar, U.J. Krull, Paper-based DNA detection using lanthanide-doped LiYF4 upconversion nanocrystals as bioprobe, Small 10 (2014) 3912–3917. [23] Q.S. Mei, H.R. Jing, Y. Li, W. Yisibashaer, J. Chen, B.N. Li, Y. Zhang, Smartphone based visual and quantitative assays on upconversional paper sensor, Biosens. Bioelectron. 75 (2016) 427–432. [24] S. Doughan, U. Uddayasankar, U.J. Krull, A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors, Anal. Chim. Acta 878 (2015) 1–8. [25] M.Y. He, Z. Li, Y.Y. Ge, Z.H. Liu, Portable upconversion nanoparticles-based paper device for field testing of drug abuse, Anal. Chem. 88 (2016) 1530–1534. [26] E. Morales-Narváez, B. Pérez-López, L.B. Pires, A. Merkoc¸i, Simple Förster resonance energy transfer evidence for the ultrahigh quantum dot quenching efficiency by graphene oxide compared to other carbon structures, Carbon 50 (2010) 2987–2993. [27] E. Morales-Narváez, A. Hassan, A. Merkoc¸i, Graphene oxide as a pathogen-revealing agent: sensing with a digital-like response, Angew. Chem. Int. Ed. 52 (2013) 13779–13783. [28] E. Morales-Narváez, T. Naghdi, E. Zor, A. Merkoc¸i, Photoluminescent lateral-flow immunoassay revealed by graphene oxide: highly sensitive paper-based pathogen detection, Anal. Chem. 87 (2015) 8573–8577. [29] H. Li, X.E. Fang, H.M. Cao, J.L. Kong, Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins, Biosens. Bioelectron. 80 (2016) 79–83. [30] B.Y. Jiang, F.Z. Li, C.Y. Yang, J.Q. Xie, Y. Xiang, R. Yuan, Aptamer pseudoknot-functionalized electronic sensor for reagentless and single-step detection of immunoglobulin E in human serum, Anal. Chem. 87 (2015) 3094–3098. [31] H.L. Li, Y.W. Zhang, L. Wang, J.Q. Tian, X.P. Sun, Nucleic acid detection using carbon nanoparticles as a fluorescent sensing platform, Chem. Commun. 47 (2011) 961–963. [32] Y.H. Wang, L. Bao, Z.H. Liu, D.W. Pang, Aptamer biosensor based on fluorescence resonance energy transfer from upconverting phosphors to carbon nanoparticles for thrombin detection in human plasma, Anal. Chem. 83 (2011) 8130–8137. [33] J.F. Nie, Y. Zhang, L.W. Lin, C.B. Zhou, S.H. Li, L.M. Zhang, J.P. Li, Low-cost fabrication of paper-based microfluidic devices by one-step plotting, Anal. Chem. 84 (2012) 6331–6335. [34] M.Y. He, Z.H. Liu, Paper-based microfluidic device with upconversion fluorescence assay, Anal. Chem. 85 (2013) 11691–11694. [35] Y.H. Wang, P. Shen, C.Y. Li, Y.Y. Wang, Z.H. Liu, Upconversion fluorescence resonance energy transfer based biosensor for ultrasensitive detection of matrix metalloproteinase-2 in blood, Anal. Chem. 84 (2012) 1466–1473.
Biographies Ping Jiang received his M.S. degree from medical college of wuhan university in 2009, and has worked in general surgery department of Zhongnan hospital of Wuhan university since then. His work covers the clinical testing of blood samples and surgical operations. Mengyuan He received her B.S. degree from Civil Aviation University of China in 2011. Then she joined Prof. Zhihong Liu’s group for graduate study. Her main research interest is in the construction of paper-based analytical devices and diagnostics. Lin Shen is currently pursuing her Master’s degree at Wuhan University. Her research interest focuses on preparation and characterization of upconversion nanoparticles. Anni Shi is an undergraduate in Wuhan University. She joined Prof. Zhihong Liu’s group in 2014. Her research interest is mainly the preparation of paper-based microfluidic analytical devices. Zhihong Liu obtained Ph. D. degree from the Department of Chemistry, Wuhan University before carrying out postdoctoral research at College of Life Science, Wuhan University and Ohio State University, USA. He is currently a professor at the Department of Chemistry, Wuhan University.