Biosensors and Bioelectronics 80 (2016) 433–441
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Molecularly imprinted plasmonic nanosensor for selective SERS detection of protein biomarkers Yongqin Lv a,n, Yating Qin a, Frantisek Svec b, Tianwei Tan a,n a b
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China International Research Center for Soft Matter, Beijing University of Chemical Technology, Beijing 100029, China
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2015 Received in revised form 29 January 2016 Accepted 31 January 2016 Available online 1 February 2016
Molecularly imprinted plasmonic nanosensor has been prepared via the rational design of an ultrathin polymer layer on the surface of gold nanorods imprinted with the target protein. This nanosensor enabled selective fishing-out of the target protein biomarker even from a complex real sample such as human serum. Sensitive SERS detection of the protein biomarkers with a strong Raman enhancement was achieved by formation of protein imprinted gold nanorods aggregates, stacking of protein imprinted gold nanorods onto a glass plate, or self-assembly of protein imprinted gold nanorods into close-packed array. High specificity and sensitivity of this method were demonstrated with a detection limit of at least 10-8 mol/L for the target protein. This could provide a promising alternative for the currently used immunoassays and fluorescence detection, and offer an ultrasensitive, non-destructive, and label-free technique for clinical diagnosis applications. & 2016 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting Gold nanorods Nanosensor Surface-enhanced Raman scattering Protein biomarker
1. Introduction Highly efficient assays that can specifically target and sensitively detect the endogenous levels of low abundance protein biomarkers are of great significance in assisting early diagnosis and prognosis of diseases, and assessing portable therapeutic response (Hanash et al., 2008; Lv et al., 2013; Mok et al., 2014). Although the immunoassays, which rely on the specific interactions between antibodies and antigens have been developed to achieve this goal, the use of antibodies has some fundamental limitations including tedious screening and production, high cost, and limited stability (Holford et al., 2012; Tothill 2009; Yang et al., 2012; Zhang et al., 2015b). Alternative synthetic artificial receptors, molecularly imprinted polymers (MIP), bear predetermined binding cavities and exhibit significant advantages consisting of ease of their preparation, low cost, high stability, and potential re-usability (G. and A. 1972; G. et al., 1973; Vlatakis et al., 1993). However, imprinting of proteins represents a major challenge that arises from their large size and multiplicity of functionalities. For example, proteins can be denatured under conventional polymerization conditions. The mass transfer of the protein within the highly crosslinked polymer networks is restricted. This represents a problem while removing the template from the polymer as well as during the n
Corresponding authors. Tel.: þ 86 10 64454356. E-mail addresses: [email protected] (Y. Lv), [email protected] (T. Tan). http://dx.doi.org/10.1016/j.bios.2016.01.092 0956-5663/& 2016 Elsevier B.V. All rights reserved.
application. Also, the binding affinity of the cavities is not homogeneous (Lv et al., 2013; Whitcombe et al., 2011). Therefore, new strategies enabling more successful design of protein imprinted nanocomposites have been developed to address these limitations. Materials prepared using these strategies facilitate mass transfer of proteins and enhance the binding capacity (Cai et al., 2010; Li et al., 2015a; Li et al., 2014a; Li et al., 2014b; Li et al., 2015b; Li et al., 2014c; Li et al., 2015c; Li et al., 2015d; Liu et al., 2015; Lv et al., 2013; Ma et al., 2015; Sykora et al., 2015; Wan et al., 2015; Zhang et al., 2015a; Zhao et al., 2014). Moreover, these nanostructured materials exhibit remarkably high surface-to-volume ratio, which enables detection of biological molecules at very low concentrations. Recently, combination of the specific recognition of MIP with surface-enhanced Raman scattering (SERS) was used for the detection of compounds at trace concentrations. For example, Bompart prepared 400 nm cross-linked core particles coated first with gold nanoparticles and then with a MIP shell. The composite significantly improved the sensitivity for the βblocking drug (S)-propranolol using detection with SERS (Bompart et al., 2010). Two groups prepared MIP on silver microspheres, and used them for SERS detection of dyes (Chang et al., 2013; Chen et al., 2014). Kamra et al. synthesized nicotineimprinted microspheres via RAFT precipitation polymerization (Kamra et al., 2015). Adopting three different approaches including direct sputtering of gold nanoparticles on the MIP surface, immobilization of gold nanoparticles through thiol functionalities of the MIP, and trapping of the MIP microspheres in a
434
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
patterned SERS substrate, they applied their systems for the detection of nicotine using SERS. Compared to fluorescence detection, SERS can provide detection limit in the femtomolar range that is about five orders of magnitude lower (Wackerlig and Lieberzeit 2015). Although fascinating, the application of this new technique remained limited mostly to the detection of metal ions and small molecules like drugs and dyes (Ben-Amram et al., 2012; Chang et al., 2013; Chen et al., 2014; Daniel et al., 2015; Feng et al., 2013; Kamra et al., 2015; Kantarovich et al., 2009, 2010; Wu et al., 2015; Xue et al., 2013; Yang et al., 2015). Recently, Ye et al. prepared imprinted boronate-affinity monolithic polymers for the determination of glycoproteins using SERS (Ye et al., 2014). Detection of glycoproteins was achieved after their fishing-out from the sample using imprinted monolithic polymer followed by labeling with boronateaffinity-functionalized silver nanoparticles in which dyes served as the SERS probe. In general, preparation of SERS-active MIPnanosensors for the specific targeting and directly sensitive detecting protein biomarkers continues to be scarce. Herein, we for the first time demonstrate the design of plasmonic MIP-nanosensors for the specific recognition and sensitive determination of protein biomarkers in complex samples using surface-enhanced Raman scattering. The MIPnanosensor was prepared by self-polymerization of dopamine on the surface of gold nanorods where the target protein was first attached. The effect of MIP layer thickness on the selective recognition and Raman enhancement was also investigated. The feasibility of this new approach is demonstrated with detection of transferrin in human serum. SERS measurements were achieved using three different approaches shown in Scheme 1: (a) formation of protein imprinted gold nanorod aggregates, (b) stacking of the protein imprinted gold nanorods on a glass plate, and (c) self-assembly of protein imprinted gold nanorods into close-packed arrays. In comparison, we also used the conventional procedure consisting in simple aggregation of gold nanorods with the target protein.
2. Experimental 2.1. Chemicals and materials Sodium borohydride (NaBH4), sodium dodecyl sulphate (SDS), dopamine hydrochloride (DA), and ascorbic acid were purchased from J&K scientific ltd (Beijing, China). silver nitrate (AgNO3), human serum albumin (HSA, pI 4.64, Mw 66 kD), myoglobin (MYO, pI 7.07, Mw 17.5 ka), lysozyme (LYZ, pI 11.0, Mw 14.0 kDa), and transferrin (TRF, pI 5.2-6.2; Mw 80 kDa) were purchased from Sigma-Aldrich (Shanghai, China). Chloroauric acid tetrahydrate (HAuCl4.4H2O) was bought from Sinopharm (Beijing, China). Cetyltrimethylammonium bromide (CTAB) was purchased from Tianjin Aoran fine chemical research institute. Tris(hydroxymethyl)aminomethane was purchased from Kemiou chemical reagent co. (Tianjin, China). All other chemicals were purchased from Beijing chemical reagent company (Beijing, China) and used as received. 2.2. Instrumentation The UV-vis spectrum was obtained from a UV-3600 UV-vis spectrophometer (Shimadzu, Japan). FTIR spectra were recorded on a Spectrum One FTIR spectrometer (Perkin Elmer, U.S.). The transmission electron microscopy (TEM) measurements were carried out using a JEOL-2100 transmission electron microscope (JEOL, USA) with an accelerating voltage of 200 kV. Elemental analysis was performed with an energy dispersive X-ray spectrometer Quantax 200 XF 5010 (Bruker, Germany). Surface enhanced Raman scattering (SERS) measurements were performed on the LabRAM ARAMIS Raman system with the 633 nm excitation laser. The spectra were recorded using an accumulation time of 15 s with a spectral resolution of 1 cm-1. 2.3. Synthesis of gold nanorods Gold nanorods (GNR) with different aspect ratios were synthesized using a seed-mediated method together with anisotropic oxidation (Ming et al., 2008; Ni et al., 2008). The seed solution was
Scheme 1. Schematic illustrations of using different approaches for SERS measurements, a) aggregation of bioconjugates, (b) stacking of bioconjugates on a glass plate, and (c) self-assembly of bioconjugates forming close-packed arrays.
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
prepared first by adding fresh and ice-cold NaBH4 aqueous solution into aqueous mixture composed of HAuCl4 and cetyltrimethylammonium bromide (CTAB), followed by rapid inversion for 2 min. This seed solution was kept at room temperature for 2 h before use. The growth solution was prepared by the sequential addition of aqueous HAuCl4 (0.01 mol/L, 2 mL), AgNO3 (0.01 mol/L, 0.4 mL), HCl (1.0 mol/L, 0.8 mL), and ascorbic acid (0.1 mol/L, 0.32 mL) in the aqueous CTAB solution (0.1 mol/L, 40 mL). After injecting the seed solution into the growth solution, the mixture was left staying overnight. Thus obtained GNR were washed several times with deionized water and concentrated by centrifugation to remove the residual CTAB from the dispersion. In order to obtain gold nanorods with different aspect ratios, the quantities of HAuCl4, CTAB, and NaBH4 used for the preparation of gold seeds, and the volume of gold seed solution added to the growth solution were adjusted as shown in Table S1 in the Supporting Information. 2.4. Protein adsorption experiments The typical procedure for protein adsorption was as follows: the MIP@GNR or NIP@GNR nanocomposite with a gold amount of 5.9 mg was added to 1 mL of standard protein solution with a concentration of 2.8 mg/mL prepared in Tris-HCl buffer (10 mmol/L, pH ¼7.5). The mixture was stirred for a period of time at 25 °C. After the adsorption process completed, the protein concentration in the supernatant was determined from the Multiskan spectrum. The adsorption capacity (Q) of MIP@GNR or NIP@GNR was calculated according to Eq. (1):
Q=
(C0 − C )V m
Q MIP Q NIP
transferrin with a concentration of 2.0 10-6 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. By adding 2.5 M NaCl and adjusting the pH value to 10, gold nanorods were aggregated with the target protein. After evaporation of water, the SERS measurement was performed on the TRF@GNR aggregates droplet. Formation of protein imprinted gold nanorods aggregates. TRFimprinted gold nanorods with a gold amount of 5.9 mg were incubated with 1 mL transferrin with a concentration of 2.0 106 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. By adding 2.5 M NaCl and adjusting the pH value to 10, TRF-imprinted gold nanorods were aggregated and measured by SERS detection. Stacking of protein imprinted gold nanorods onto a glass plate. TRF-imprinted gold nanorods with a gold amount of 5.9 mg were incubated with 1 mL transferrin with a concentration of 2.0 10-6 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. One drop of the MIP@GNR solution was dripped on the surface of a glass plate. After 5 min evaporation of the water, the substrate was placed on the stage of the Raman system for analysis. Self-assembly of protein imprinted gold nanorods into closepacked arrays. The preparation of protein imprinted gold nanorods into close-packed arrays was followed previously reported work with some modifications (Cecchini et al., 2013). TRF-imprinted gold nanorods with a gold amount of 5.9 mg were incubated with 1 mL transferrin with a concentration of 2.0 10-6 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. Then, 200 μL of the above MIP@GNR dispersion was dissolved in 1 mL 1,2-dichloroethane. After stirring for 5 min, the W/O microspheres were generated. The SERS measurement was performed on the droplet of the W/O microspheres after evaporation of the water phase.
(1)
where C0 is the initial concentration of protein (mg/mL), C is the final protein concentration in the supernatant (mg/mL), V is the volume of solution (mL), and m (mg) is the dry weight of MIP@GNR or NIP@GNR nanocomposites in each adsorption solution. The imprinting factor (IF, α) was used to evaluate the specificity and selectivity of the prepared imprinted nanocomposites (Eq. 2):
α=
435
(2)
where QMIP and QNIP are the adsorption capacities of MIP@GNR and NIP@GNR nanocomposites for the template protein, respectively. 2.5. Preparation of the MIP@GNR and NIP@GNR nanocomposites The preparations of TRF-MIP@GNR and NIP@GNR nanocomposites were as follows. At first, 10 mg transferrin (TRF) was dissolved in 3.5 mL 10 mM Tris-HCl buffer (pH 7.5), to which 5.9 mg gold nanorods was added. After stirring for 2 h, 7 mg dopamine hydrochloride (DA) was added into the solution. The mixture was then vigorously stirred at room temperature for 8 h. After the reaction, the resultant TRF-MIP@GNR was washed with deionized water to remove unreacted transferrin and dopamine. The imprinted transferrin was then removed using 10% (w/v) SDS solution, followed by further washing the TRF-MIP@GNR nanocomposites with deionized water. For comparison, the NIP@GNR was prepared using the same synthetic approach in the absence of transferrin template. The synthesis of human serum albumin (HSA) imprinted gold nanorods followed the same procedure as the one designed for TRF-MIP@GNR. 2.6. SERS detection Aggregation of gold nanorods with the target protein. Gold nanorods with an amount of 5.9 mg were incubated with 1 mL
3. Results and discussion 3.1. Design of molecularly imprinted plasmonic nanosensors Gold nanorods exhibit transverse and longitudinal surface plasmon resonances that are deemed to be more SERS-active and to enable stronger Raman enhancement compared with spherical gold nanoparticles (Ni et al., 2008; Nikoobakht and El-Sayed, 2003). In this study, the gold nanorods (GNR) were prepared using the seed-mediated method together with anisotropic oxidation according to previous reports (Ming et al., 2008; Ni et al., 2008). Transmission electron microscopy (TEM) image shown in Fig. 1a confirms the uniform size of GNR with an average diameter of 267 2 nm and length of 8075 nm representing an aspect ratio (the length to width ratio) of 3.1 70.4 nm. The UV-vis spectrum of GNR dispersion is shown in Fig. 1b. The longitudinal surface plasmon resonance (LSPR) peak centered at 511 nm corresponds to the initial gold nanoparticle seeds. A new peak with stronger intensity appears at 775 nm and proves the growth of gold nanorods. Transferrin (TRF, pI 5.2-6.2; molecular weight 77.0 kDa), which was used as the template molecule, is iron-binding blood plasma glycoprotein. It is regarded as a protein biomarker that controls the level of free iron in biological fluids (Crichton and CharloteauxWauters, 1987). This protein was first adsorbed at the surface of the GNR via electrostatic interactions, and then the polydopamine (PDA) thin layer was prepared at the surface of GNR with attached transferrins via the self-polymerization of dopamine. The embedded protein templates were extracted with 10% (w/v) sodium dodecyl sulphate solution for 5 min to produce the molecularly imprinted layer. The polymer layer at the MIP@GNR core-shell structure had a thickness of 5.3 70.4 nm (Fig. 1c). A thin layer is favorable since it facilitates rapid mass transfer during protein adsorption and desorption. For comparison, non-imprinted
436
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
Fig. 1. TEM image of gold nanorods (a), UV-vis spectrum of gold nanorods dispersion (b), TEM image of transferrin-imprinted gold nanorods (c), FTIR spectra of nonimprinted (black line) and transferrin-imprinted gold nanorods (red line) (d), and XRD patterns of gold nanorods (red line), their counterparts coated with a thin layer of polydopamine (blue line), and the simulated pattern (bottom) (e).
polymer (NIP) thin layer was also prepared on the surface of gold nanorods using the same conditions except for the absence of protein templates. Energy-dispersive X-ray spectroscopy revealed 8.9 wt% nitrogen for MIP@GNR which demonstrated the successful coating of GNR with polydopamine layer. FTIR spectra of MIP@GNR and NIP@GNR nanocomposites shown in Fig. 1d exhibit peaks centered at 3430, 3145, 1636, and 1400 cm-1 ascribed to the stretching vibrations of hydroxyl, amine, C-N, and phenyl groups of polydopamine layer. Further characterizations of GNR and MIP@GNR were achieved using X-ray diffraction (XRD) patterns (Fig. 1e) that feature diffraction peaks typical of gold nanorods according to the JCPDS card (No. 04-0784). 3.2. Rebinding performances of protein-imprinted gold nanorods Both TRF-imprinted and non-imprinted gold nanorods were incubated in a transferrin solution. Their adsorption kinetic curves were presented in Fig. 2a. While the NIP@GNR binding capacity for transferrin was 16.7 mg/g, 66.6 mg/g of transferrin was found for MIP@GNR. The adsorption of transferrin on NIP@GNR originated merely from the non-specific binding. In contrast, imprinting significantly enlarged the adsorption capacity of MIP@GNR and resulted in an imprinting factor of 4.0. Thanks to the small thickness of the polymer layers, the equilibrium adsorption was achieved in a remarkably short time of only 7 min for NIP@GNR and 15 min for MIP@GNR. The slightly faster binding kinetics on NIP can be attributed to the easy diffusion to and fast interaction of transferrin with the non-specific sites located at the polymer surface. The adsorption capacity and adsorption/desorption kinetics of our imprinted nanocomposites are comparable or even exceed those
observed by other authors for the core-shell MIP nanosystems (Gao et al., 2011; Li et al., 2015d; Wan et al., 2015; Zhang et al., 2010; Zhang et al., 2012). The rebinding specificity and selectivity of the TRF-imprinted GNR was evaluated using adsorption capacities for the target protein and potentially interfering blood plasma proteins including myoglobin (pI 7.07; molecular weight 17.5 kDa), lysozyme (pI 11.0; molecular weight 14.3 kDa), and human serum albumin (pI 4.64; molecular weight 66.0 kDa). Fig. 2b confirms similar affinity for all four proteins on the non-imprinted surface. In contrast, the TRF-imprinted surface exhibited excellent selectivity towards its own template protein transferrin compared to a significantly lower affinity for other proteins. The imprinting factors (IF) of TRF-imprinted surface for myoglobin, lysozyme, and human serum albumin were only 1.2, 1.4, and 1.2, respectively. To investigate the general feasibility of our imprinting approach, human serum albumin was also used as the template molecule to prepare HSA-imprinted PDA@GNR nanosensor via the same synthetic procedure of self-polymerization. Similar to the transferrin imprinted system, the HSA-MIP@GNR exhibited a large adsorption capacity of 92.4 mg/g for human serum albumin that compared favorably to the value of 23.7 mg/g observed on the NIP (Fig. 2c). Binding experiments using other proteins on both MIP and NIP confirmed the high selectivity and specificity of the HSAimprinted polymer layer, which has imprinting factors of 3.9, 1.1, 1.9, and 1.5 for the template protein, myoglobin, lysozyme, and transferrin, respectively.
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
437
Fig. 2. The rebinding curves of the TRF-imprinted and non-imprinted polydopamine layers towards transferrin (a), the adsorption capacities of the TRF-imprinted, and nonimprinted polydopamine layers towards transferrin (TRF), myoglobin (MYO), lysozyme (LYZ), and human serum albumin (HSA) (b), and the adsorption capacities of the HSAimprinted, and non-imprinted polydopamine layers towards human serum albumin (HSA), myoglobin (MYO), lysozyme (LYZ), and transferrin (TRF) (c). (Protein concentration, 2.86 mg/mL).
3.3. SERS performances To study the performances of the TRF-imprinted nanosensors in SERS, both MIP@GNR and NIP@GNR were first incubated with transferrin solutions having a concentration of 2.0 10-6 mol/L to reach their adsorption equilibrium. In this way, the imprinted cavities of MIP@GNR were filled with transferrin. Then 2.5 mol/L of NaCl solution with pH value adjusted to 10 was added to initiate the aggregation of the nanorods and formation of “hot spots” (Scheme 1a). Raman spectra of TRF@MIP@GNR and TRF@NIP@GNR aggregates presented in Fig. 3a show that spectrum of the imprinted nanosensor incubated in transferrin is significantly enhanced. The characteristic peaks can be assigned to the vibrations of amide I (1626 cm-1) and amide III (1221 cm1 ) of α-helix structure, C-O and C-N stretching vibrations (1077 cm-1), C-C vibrations (952 cm-1), COO- vibrations (1399 cm-1), Cys (457 cm-1), Met (726 cm-1), Ala (892, 952 cm-1), and aromatic residues of Trp (590, 1531 cm-1), and Tyr (843 cm-1) of transferrin. In contrast, the enhancements in SERS spectra acquired for transferrin applied to both aggregated NIP@GNR and gold nanorod colloids were very weak. In all spectra, the GNR background is characterized with peaks centered at 761 and 1448 cm-1. The large difference in signal strength clearly results from the presence of specific binding sites contained in the imprinted polymer shell that selectively adsorb the protein. In addition to the above experiments using TRF@MIP@GNR aggregates, the SERS determination of transferrin was also carried out with samples prepared using simple application of the transferrin bioconjugate on a glass plate (Scheme 1b) as well as using the procedure reported by Cecchini et al. (Cecchini et al., 2013) that leads to self-assembled TRF@MIP@GNR in close-packed arrays at the liquid/liquid interface (Scheme 1c). The spectra are shown in Fig. 3b. The SERS spectrum acquired from sample at the glass plate
exhibited strong signals although with a reduced number of prominent peaks observed for C-C and C-N stretching vibrations (952 and 1123 cm-1), COO- vibrations (1362 cm-1), and Phe (1576 cm-1). The close-packed arrays featured more characteristic peaks attributed to Cys (469 cm-1), amide I (1629 cm-1) and amide III (1206 cm-1), C-C and C-N stretching vibrations (956, 1063 cm-1), Phe (1562 cm-1), Trp (1504 cm-1), and Tyr (657, 835 cm-1) but with significantly weaker intensities compared to those observed for aggregated TRF@MIP@GNR conjugate. The TRF-imprinted polydopamine gold nanorods were also incubated in transferrin solutions with low concentrations in order to elucidate the limits of sensitivity enabled by the MIP shell. Fig. 3c illustrates the Raman spectra of transferrin monitored in a concentration range of 5.6 10-8 mol/L to 2.1 10-6 mol/L confirming the appearance of identifiable Raman peaks even at a concentration as low as 5.6 10-8 mol/L. The insert in the Figure demonstrates a linear relationship between the integrated intensity of the peaks centered at 1395 cm-1 and the concentration of transferrin. These results also indicate that the detection limit of our imprinted plasmonic nanosensors is 10-8 mol/L. Identically, to demonstrate the versatility of our imprinting/SERS method, the HSA-imprinted polydopamine gold nanorods were incubated with 2.6 10-6 mol/L human serum albumin, the aggregated HSA@PDA@GNR bioconjugates were applied for the SERS detection. The SERS spectrum of human serum albumin presented in Fig. 4 (a) shows vibrations of amide I (1637 cm-1) and amide III (1250 cm-1) bands due to the α-helix structure, C-N stretching vibrations (1086 cm-1), COO- vibrations (1389 cm-1), Met (719 cm-1), Val (925 cm-1), Ala (951 cm-1), and aromatic residues such as Phe (1025 and 1580 cm-1), Tyr (669 and 841 cm-1), and Trp (561, 753, 1180, and 1543 cm-1) that are typical of human serum albumin.
438
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
Fig. 3. a) SERS spectra of gold nanorods (GNRs), TRF@GNR aggregates, TRF@NIP@GNR aggregates, and TRF@MIP@GNR aggregates (transferrin concentration, 2.0 10-6 mol/ L); b) SERS spectra of protein imprinted gold nanorods stacked onto a glass plate, and self-assembly into close-packed arrays after incubation with 2.0 10-6 mol/L transferrin; c) SERS spectra of transferrin determined on TFR@MIP@GNR aggregates with different concentrations ranging from 2.1 10-6 mol/L to 5.6 10-8 mol/L; d) SERS spectra of TFR@MIP@GNR aggregates with different thickness of polydopamine layer prepared from dopamine monomer solutions with concentrations ranging from 0.5 to 4.0 mg/mL (transferrin concentration, 2.0 10-6 mol/L); and e) SERS spectra of TFR@MIP@GNR aggregates prepared from nanorods with different aspect ratios (transferrin concentration, 2.0 10-6 mol/L).
3.4. Effects of the thickness of molecularly imprinted polymer layer on SERS detection The thickness of molecularly imprinted polymer shell is important for the function of the sensor. It is known that the thickness of the MIP shell needs to be preferably less than one particle
radius to guarantee that the target molecule is in the close vicinity of gold nanorods and to achieve the largest enhancement of the SERS signal (Bompart et al., 2010). A thinner layer facilitates mass transfer to and from the imprinted sites. However, the number of the available sites is low and the overall loading capacity is limited. Also, the imprinted cavities in a too thin layer may not have the
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
439
Fig. 4. a) SERS spectra of HSA-imprinted gold nanorods bioconjugates after incubation with 2.6 10-6 mol/L human serum albumin, and b) SERS spectra of TRF-imprinted gold nanorods bioconjugates after incubation with 2.0 10-6 mol/L transferrin in human serum.
perfect shape. Opposite applies to the thick layer. With the increase of layer thickness, the distance between the aggregated GNR is increased, and the probability of formation of “hot spots” that enhance the signal will decrease rapidly. Therefore, an optimum layer thickness must be found. The thickness of the layer is controlled by the concentration of dopamine in the monomer solution. In our experiments, we used concentrations ranging from 0.5 to 4.0 mg/mL. As expected, a high dopamine concentration of 4.0 mg/mL generates a 8.7 70.5 nm thick polydopamine layer on the surface of gold nanorods as estimated from TEM images shown in Fig. S1 in the Supporting Information. In contrast, Fig. 1c demonstrates that the polymer layer thickness was 5.3 70.4 nm while using 2.0 mg/mL dopamine. When the concentration of dopamine further decreased to 1.0 and 0.5 mg/mL, the polymer layer thickness on the surface of gold nanorods was 3.5 70.3 and 1.270.3 nm, respectively (Fig. S1). Our MIP@GNR bioconjugates differing in thickness of polydopamine layer and incubated with 2.0 10-6 mol/L transferrin solution were aggregated, and then used for the SERS detection. Fig. 3d illustrates that the nanosensor prepared using 2.0 mg/mL dopamine solution provided the most enhanced Raman scattering intensity. 3.5. Effect of aspect ratio of gold nanorods The use of gold nanorods with different aspect ratios enables tuning of the surface plasmon band to the wavelength of excitation laser to obtain the largest enhancement. Therefore, effects of aspect ratio of GNR on Raman enhancement were also studied and a series of gold nanorods with different aspect ratios were prepared. The transmission electron microscopy images shown in Fig. 5 confirm the successful preparation of nanorods with aspect ratio of 4.3 7 0.4, 3.4 70.3, and 2.9 70.3, respectively. The TRF@MIP@GNR aggregates were then subjected to Raman scattering measurements. Fig. 3(e) illustrates that the TFR@MIP@GNR aggregates prepared from nanorods with an aspect ratio of 2.9 70.3 exhibited the strongest Raman enhancement. 3.6. Practical application using real-life sample We also tested the performance of our imprinted plasmonic nanosensors in the detection of protein biomarker in real biological samples. The TRF-imprinted PDA@GNR was incubated with human serum spiked with 2 10-6 mol/L transferrin. After incubation, the nanocomposites were collected by centrifugation,
washed with Tris-HCl buffer, and the bound transferrin was detected by Raman spectroscopy. The Raman spectrum in Fig. 4b shows prominent peaks centered at 453 cm-1, 729 cm-1, 835 cm-1, 960 cm-1, 1060 cm-1, 1202 cm-1, 1395 cm-1, and 1536 cm-1 that are attributed to transferrin. Besides, we also observed some very weak characteristic bands at 567 cm-1, 673 cm-1, and 1270 cm-1 which might be ascribed to human serum albumin due to the nonspecific adsorption. A relative standard deviation of 8.4% was calculated from measurements of three parallel human sera, and the recovery was 98.6%. This result confirmed that our imprinted nanosensor has both high selectivity and specificity for the trace concentration of target protein even if used with real biological samples, and indicated a great potential and practical applicability of our approach in the detection of protein biomarkers applicable for clinical diagnostics.
4. Conclusions We have presented the rational design of new plasmonic imprinted nanosensors, which were applied for the selective “fishing-out” and sensitive SERS detection of the target protein from both aqueous solutions and human serum samples. Competitive rebinding studies with potentially interfering proteins and real life serum sample demonstrated that the imprinted polymer layer had a high selectivity for the targeted protein biomarker. Formation of the TRF@MIP@GNR aggregates enabled SERS detection of the protein presented at a concentration of at least 10-8 mol/L. Our system appears to be a promising tool for the early diagnosis and prognosis of diseases that are manifested with protein biomarkers.
Acknowledgment The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (21576017, 21436002, and 21306006), 973 programs (2014CB745103, 2013CB733603), 863 programs (2015BAD15B07), the Xiamen Scientific and Technological project (3502Z20142012), the Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology, and the Fundamental Research Funds for the Central Universities (YS1407, buctrc201413).
440
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
Fig. 5. TEM images of gold nanorods with different aspect ratios of 4.3 7 0.4 (a, b), 3.4 70.4 (c, d), and 2.9 7 0.3 (e, f).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.01.092.
References Ben-Amram, Y., Tel-Vered, R., Riskin, M., Wang, Z.G., Willner, I., 2012. Chem. Sci. 3, 162–167. Bompart, M., De Wilde, Y., Haupt, K., 2010. Adv. Mater. 22, 2343–2348.
Cai, D., Ren, L., Zhao, H., Xu, C., Zhang, L., Yu, Y., Wang, H., Lan, Y., Roberts, M.F., Chuang, J.H., Naughton, M.J., Ren, Z., Chiles, T.C., 2010. Nat. Nanotechnol. 5, 597–601. Cecchini, M.P., Turek, V.A., Paget, J., Kornyshev, A.A., Edel, J.B., 2013. Nat. Mater. 12, 165–171. Chang, L., Ding, Y., Li, X., 2013. Biosens. Bioelectron. 50, 106–110. Chen, S., Li, X., Zhao, Y., Chang, L., Qi, J., 2014. Chem. Commun. 50, 14331–14333. Crichton, R.R., Charloteaux-Wauters, M., 1987. Eur. J. Biochem. 164, 485–506. Daniel, S., Matikainen, A., Turunen, J., Vahimaa, P., Nuutinen, T., 2015. J. Colloid Interface Sci. 437, 119–123. Feng, S., Gao, F., Chen, Z., Grant, E., Kitts, D.D., Wang, S., Lu, X., 2013. J. Agric. Food. Chem. 61, 10467–10475. G, W., A, S., 1972. Angew. Chem. Int. Ed. 11, 341–348.
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
G, W., A, S., K, Z., 1973. Tetrahedron Lett. 44, 4329–4332. Gao, R., Kong, X., Wang, X., He, X., Chen, L., Zhang, Y., 2011. J. Mater. Chem. 21, 17863–17871. Hanash, S.M., Pitteri, S.J., Faca, V.M., 2008. Nature 452, 571–579. Holford, T.R.J., Davis, F., Higson, S.P.J., 2012. Biosens. Bioelectron. 34, 12–24. Kamra, T., Zhou, T., Montelius, L., Schnadt, J., Ye, L., 2015. Anal. Chem. 87, 5056–5061. Kantarovich, K., Tsarfati, I., Gheber, L.A., Haupt, K., Bar, I., 2009. Anal. Chem. 81, 5686–5690. Kantarovich, K., Tsarfati, I., Gheber, L.A., Haupt, K., Bar, I., 2010. Biosens. Bioelectron. 26, 809–814. Li, D.Y., Qin, Y.P., Li, H.Y., He, X.W., Li, W.Y., Zhang, Y.K., 2015a. Biosens. Bioelectron. 66, 224–230. Li, N., Qi, L., Shen, Y., Qiao, J., Chen, Y., 2014a. ACS Appl. Mater. Inter. 6, 17289–17295. Li, Q., Yang, K., Liang, Y., Jiang, B., Liu, J., Zhang, L., Liang, Z., Zhang, Y., 2014b. ACS Appl. Mater. Inter. 6, 21954–21960. Li, S., Yang, K., Liu, J., Jiang, B., Zhang, L., Zhang, Y., 2015b. Anal. Chem. 87, 4617–4620. Li, X., Zhang, B., Li, W., Lei, X., Fan, X., Tian, L., Zhang, H., Zhang, Q., 2014c. Biosens. Bioelectron. 51, 261–267. Li, X., Zhang, B., Tian, L., Li, W., Zhang, H., Zhang, Q., 2015c. Sens. Actuators, B 208, 559–568. Li, Y., Bin, Q., Lin, Z., Chen, Y., Yang, H., Cai, Z., Chen, G., 2015d. Chem. Commun 51, 202–205. Liu, Y., Fang, S., Zhai, J., Zhao, M., 2015. Nanoscale 7, 7162–7167. Lv, Y., Tan, T., Svec, F., 2013. Biotechnol. Adv. 31, 1172–1186. Ma, Y., Xu, S., Wang, S., Wang, L., 2015. TrAC, Trends Anal. Chem. 67, 209–216. Ming, T., Kou, X., Chen, H., Wang, T., Tam, H.L., Cheah, K.W., Chen, J.Y., Wang, J., 2008. Angew. Chem. Int. Ed. 47, 9685–9690. Mok, J., Mindrinos, M.N., Davis, R.W., Mehdi, J., 2014. PNAS. 111, 2110–2115.
441
Ni, W., Kou, X., Yang, Z., Wang, J., 2008. ACS Nano. 2, 677–686. Nikoobakht, B., El-Sayed, M.A., 2003. Chem. Mater. 15, 1957–1962. Sykora, S., Cumbo, A., Belliot, G., Pothier, P., Arnal, C., Dudal, Y., Corvini, P.F.X., Shahgaldian, P., 2015. Chem. Commun. 51, 2256–2258. Tothill, I.E., 2009. Semin. Cell Dev. Biol. 20, 55–62. Vlatakis, G., Andersson, L.I., Muller, R., Mosbach, K., 1993. Nature 361, 645–647. Wackerlig, J., Lieberzeit, P.A., 2015. Sens. Actuators, B 207, 144–157. Wan, W., Han, Q., Zhang, X., Xie, Y., Sun, J., Ding, M., 2015. Chem. Commun. 51, 3541–3544. Whitcombe, M.J., Chianella, I., Larcombe, L., Piletsky, S.A., Noble, J., Porter, R., Horgan, A., 2011. Chem. Soc. Rev. 40, 1547–1571. Wu, Y.Y., Yang, C.X., Yan, X.P., 2015. Analyst. 140, 3107–3112. Xue, J.Q., Li, D.W., Qu, L.L., Long, Y.T., 2013. Anal. Chim. Acta 777, 57–62. Yang, J.R., Xie, S.M., Liu, H., Zhang, J.H., Yuan, L.M., 2015. Chromatographia 78, 557–564. Yang, K., Zhang, L., Liang, Z., Zhang, Y., 2012. Anal. Bioanal. Chem. 403, 2173–2183. Ye, J., Chen, Y., Liu, Z., 2014. Angew. Chem. Int. Ed. 53, 10386–10389. Zhang, M., Huang, J., Yu, P., Chen, X., 2010. Talanta 81, 162–166. Zhang, S.R., Li, J., Du, D.Y., Qin, J.S., Li, S.L., He, W.W., Su, Z.M., Lan, Y.Q., 2015a. J. Mater. Chem. A 3, 23426–23434. Zhang, W., He, X.W., Chen, Y., Li, W.Y., Zhang, Y.K., 2012. Biosens. Bioelectron. 31, 84–89. Zhang, Z., Guan, Y., Li, M., Zhao, A., Ren, J., Qu, X., 2015b. Chem. Sci. 6, 2822–2826. Zhao, X.L., Li, D.Y., He, X.W., Li, W.Y., Zhang, Y.K., 2014. J. Mater. Chem. B 2, 7575–7582.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Molecularly imprinted plasmonic nanosensor for selective SERS detection of protein biomarkers Yongqin Lv a,n, Yating Qin a, Frantisek Svec b, Tianwei Tan a,n a b
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China International Research Center for Soft Matter, Beijing University of Chemical Technology, Beijing 100029, China
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2015 Received in revised form 29 January 2016 Accepted 31 January 2016 Available online 1 February 2016
Molecularly imprinted plasmonic nanosensor has been prepared via the rational design of an ultrathin polymer layer on the surface of gold nanorods imprinted with the target protein. This nanosensor enabled selective fishing-out of the target protein biomarker even from a complex real sample such as human serum. Sensitive SERS detection of the protein biomarkers with a strong Raman enhancement was achieved by formation of protein imprinted gold nanorods aggregates, stacking of protein imprinted gold nanorods onto a glass plate, or self-assembly of protein imprinted gold nanorods into close-packed array. High specificity and sensitivity of this method were demonstrated with a detection limit of at least 10-8 mol/L for the target protein. This could provide a promising alternative for the currently used immunoassays and fluorescence detection, and offer an ultrasensitive, non-destructive, and label-free technique for clinical diagnosis applications. & 2016 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting Gold nanorods Nanosensor Surface-enhanced Raman scattering Protein biomarker
1. Introduction Highly efficient assays that can specifically target and sensitively detect the endogenous levels of low abundance protein biomarkers are of great significance in assisting early diagnosis and prognosis of diseases, and assessing portable therapeutic response (Hanash et al., 2008; Lv et al., 2013; Mok et al., 2014). Although the immunoassays, which rely on the specific interactions between antibodies and antigens have been developed to achieve this goal, the use of antibodies has some fundamental limitations including tedious screening and production, high cost, and limited stability (Holford et al., 2012; Tothill 2009; Yang et al., 2012; Zhang et al., 2015b). Alternative synthetic artificial receptors, molecularly imprinted polymers (MIP), bear predetermined binding cavities and exhibit significant advantages consisting of ease of their preparation, low cost, high stability, and potential re-usability (G. and A. 1972; G. et al., 1973; Vlatakis et al., 1993). However, imprinting of proteins represents a major challenge that arises from their large size and multiplicity of functionalities. For example, proteins can be denatured under conventional polymerization conditions. The mass transfer of the protein within the highly crosslinked polymer networks is restricted. This represents a problem while removing the template from the polymer as well as during the n
Corresponding authors. Tel.: þ 86 10 64454356. E-mail addresses: [email protected] (Y. Lv), [email protected] (T. Tan). http://dx.doi.org/10.1016/j.bios.2016.01.092 0956-5663/& 2016 Elsevier B.V. All rights reserved.
application. Also, the binding affinity of the cavities is not homogeneous (Lv et al., 2013; Whitcombe et al., 2011). Therefore, new strategies enabling more successful design of protein imprinted nanocomposites have been developed to address these limitations. Materials prepared using these strategies facilitate mass transfer of proteins and enhance the binding capacity (Cai et al., 2010; Li et al., 2015a; Li et al., 2014a; Li et al., 2014b; Li et al., 2015b; Li et al., 2014c; Li et al., 2015c; Li et al., 2015d; Liu et al., 2015; Lv et al., 2013; Ma et al., 2015; Sykora et al., 2015; Wan et al., 2015; Zhang et al., 2015a; Zhao et al., 2014). Moreover, these nanostructured materials exhibit remarkably high surface-to-volume ratio, which enables detection of biological molecules at very low concentrations. Recently, combination of the specific recognition of MIP with surface-enhanced Raman scattering (SERS) was used for the detection of compounds at trace concentrations. For example, Bompart prepared 400 nm cross-linked core particles coated first with gold nanoparticles and then with a MIP shell. The composite significantly improved the sensitivity for the βblocking drug (S)-propranolol using detection with SERS (Bompart et al., 2010). Two groups prepared MIP on silver microspheres, and used them for SERS detection of dyes (Chang et al., 2013; Chen et al., 2014). Kamra et al. synthesized nicotineimprinted microspheres via RAFT precipitation polymerization (Kamra et al., 2015). Adopting three different approaches including direct sputtering of gold nanoparticles on the MIP surface, immobilization of gold nanoparticles through thiol functionalities of the MIP, and trapping of the MIP microspheres in a
434
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
patterned SERS substrate, they applied their systems for the detection of nicotine using SERS. Compared to fluorescence detection, SERS can provide detection limit in the femtomolar range that is about five orders of magnitude lower (Wackerlig and Lieberzeit 2015). Although fascinating, the application of this new technique remained limited mostly to the detection of metal ions and small molecules like drugs and dyes (Ben-Amram et al., 2012; Chang et al., 2013; Chen et al., 2014; Daniel et al., 2015; Feng et al., 2013; Kamra et al., 2015; Kantarovich et al., 2009, 2010; Wu et al., 2015; Xue et al., 2013; Yang et al., 2015). Recently, Ye et al. prepared imprinted boronate-affinity monolithic polymers for the determination of glycoproteins using SERS (Ye et al., 2014). Detection of glycoproteins was achieved after their fishing-out from the sample using imprinted monolithic polymer followed by labeling with boronateaffinity-functionalized silver nanoparticles in which dyes served as the SERS probe. In general, preparation of SERS-active MIPnanosensors for the specific targeting and directly sensitive detecting protein biomarkers continues to be scarce. Herein, we for the first time demonstrate the design of plasmonic MIP-nanosensors for the specific recognition and sensitive determination of protein biomarkers in complex samples using surface-enhanced Raman scattering. The MIPnanosensor was prepared by self-polymerization of dopamine on the surface of gold nanorods where the target protein was first attached. The effect of MIP layer thickness on the selective recognition and Raman enhancement was also investigated. The feasibility of this new approach is demonstrated with detection of transferrin in human serum. SERS measurements were achieved using three different approaches shown in Scheme 1: (a) formation of protein imprinted gold nanorod aggregates, (b) stacking of the protein imprinted gold nanorods on a glass plate, and (c) self-assembly of protein imprinted gold nanorods into close-packed arrays. In comparison, we also used the conventional procedure consisting in simple aggregation of gold nanorods with the target protein.
2. Experimental 2.1. Chemicals and materials Sodium borohydride (NaBH4), sodium dodecyl sulphate (SDS), dopamine hydrochloride (DA), and ascorbic acid were purchased from J&K scientific ltd (Beijing, China). silver nitrate (AgNO3), human serum albumin (HSA, pI 4.64, Mw 66 kD), myoglobin (MYO, pI 7.07, Mw 17.5 ka), lysozyme (LYZ, pI 11.0, Mw 14.0 kDa), and transferrin (TRF, pI 5.2-6.2; Mw 80 kDa) were purchased from Sigma-Aldrich (Shanghai, China). Chloroauric acid tetrahydrate (HAuCl4.4H2O) was bought from Sinopharm (Beijing, China). Cetyltrimethylammonium bromide (CTAB) was purchased from Tianjin Aoran fine chemical research institute. Tris(hydroxymethyl)aminomethane was purchased from Kemiou chemical reagent co. (Tianjin, China). All other chemicals were purchased from Beijing chemical reagent company (Beijing, China) and used as received. 2.2. Instrumentation The UV-vis spectrum was obtained from a UV-3600 UV-vis spectrophometer (Shimadzu, Japan). FTIR spectra were recorded on a Spectrum One FTIR spectrometer (Perkin Elmer, U.S.). The transmission electron microscopy (TEM) measurements were carried out using a JEOL-2100 transmission electron microscope (JEOL, USA) with an accelerating voltage of 200 kV. Elemental analysis was performed with an energy dispersive X-ray spectrometer Quantax 200 XF 5010 (Bruker, Germany). Surface enhanced Raman scattering (SERS) measurements were performed on the LabRAM ARAMIS Raman system with the 633 nm excitation laser. The spectra were recorded using an accumulation time of 15 s with a spectral resolution of 1 cm-1. 2.3. Synthesis of gold nanorods Gold nanorods (GNR) with different aspect ratios were synthesized using a seed-mediated method together with anisotropic oxidation (Ming et al., 2008; Ni et al., 2008). The seed solution was
Scheme 1. Schematic illustrations of using different approaches for SERS measurements, a) aggregation of bioconjugates, (b) stacking of bioconjugates on a glass plate, and (c) self-assembly of bioconjugates forming close-packed arrays.
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
prepared first by adding fresh and ice-cold NaBH4 aqueous solution into aqueous mixture composed of HAuCl4 and cetyltrimethylammonium bromide (CTAB), followed by rapid inversion for 2 min. This seed solution was kept at room temperature for 2 h before use. The growth solution was prepared by the sequential addition of aqueous HAuCl4 (0.01 mol/L, 2 mL), AgNO3 (0.01 mol/L, 0.4 mL), HCl (1.0 mol/L, 0.8 mL), and ascorbic acid (0.1 mol/L, 0.32 mL) in the aqueous CTAB solution (0.1 mol/L, 40 mL). After injecting the seed solution into the growth solution, the mixture was left staying overnight. Thus obtained GNR were washed several times with deionized water and concentrated by centrifugation to remove the residual CTAB from the dispersion. In order to obtain gold nanorods with different aspect ratios, the quantities of HAuCl4, CTAB, and NaBH4 used for the preparation of gold seeds, and the volume of gold seed solution added to the growth solution were adjusted as shown in Table S1 in the Supporting Information. 2.4. Protein adsorption experiments The typical procedure for protein adsorption was as follows: the MIP@GNR or NIP@GNR nanocomposite with a gold amount of 5.9 mg was added to 1 mL of standard protein solution with a concentration of 2.8 mg/mL prepared in Tris-HCl buffer (10 mmol/L, pH ¼7.5). The mixture was stirred for a period of time at 25 °C. After the adsorption process completed, the protein concentration in the supernatant was determined from the Multiskan spectrum. The adsorption capacity (Q) of MIP@GNR or NIP@GNR was calculated according to Eq. (1):
Q=
(C0 − C )V m
Q MIP Q NIP
transferrin with a concentration of 2.0 10-6 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. By adding 2.5 M NaCl and adjusting the pH value to 10, gold nanorods were aggregated with the target protein. After evaporation of water, the SERS measurement was performed on the TRF@GNR aggregates droplet. Formation of protein imprinted gold nanorods aggregates. TRFimprinted gold nanorods with a gold amount of 5.9 mg were incubated with 1 mL transferrin with a concentration of 2.0 106 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. By adding 2.5 M NaCl and adjusting the pH value to 10, TRF-imprinted gold nanorods were aggregated and measured by SERS detection. Stacking of protein imprinted gold nanorods onto a glass plate. TRF-imprinted gold nanorods with a gold amount of 5.9 mg were incubated with 1 mL transferrin with a concentration of 2.0 10-6 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. One drop of the MIP@GNR solution was dripped on the surface of a glass plate. After 5 min evaporation of the water, the substrate was placed on the stage of the Raman system for analysis. Self-assembly of protein imprinted gold nanorods into closepacked arrays. The preparation of protein imprinted gold nanorods into close-packed arrays was followed previously reported work with some modifications (Cecchini et al., 2013). TRF-imprinted gold nanorods with a gold amount of 5.9 mg were incubated with 1 mL transferrin with a concentration of 2.0 10-6 mol/L dissolving in Tris-HCl buffer (10 mM, pH 7.5) for 15 min. Then, 200 μL of the above MIP@GNR dispersion was dissolved in 1 mL 1,2-dichloroethane. After stirring for 5 min, the W/O microspheres were generated. The SERS measurement was performed on the droplet of the W/O microspheres after evaporation of the water phase.
(1)
where C0 is the initial concentration of protein (mg/mL), C is the final protein concentration in the supernatant (mg/mL), V is the volume of solution (mL), and m (mg) is the dry weight of MIP@GNR or NIP@GNR nanocomposites in each adsorption solution. The imprinting factor (IF, α) was used to evaluate the specificity and selectivity of the prepared imprinted nanocomposites (Eq. 2):
α=
435
(2)
where QMIP and QNIP are the adsorption capacities of MIP@GNR and NIP@GNR nanocomposites for the template protein, respectively. 2.5. Preparation of the MIP@GNR and NIP@GNR nanocomposites The preparations of TRF-MIP@GNR and NIP@GNR nanocomposites were as follows. At first, 10 mg transferrin (TRF) was dissolved in 3.5 mL 10 mM Tris-HCl buffer (pH 7.5), to which 5.9 mg gold nanorods was added. After stirring for 2 h, 7 mg dopamine hydrochloride (DA) was added into the solution. The mixture was then vigorously stirred at room temperature for 8 h. After the reaction, the resultant TRF-MIP@GNR was washed with deionized water to remove unreacted transferrin and dopamine. The imprinted transferrin was then removed using 10% (w/v) SDS solution, followed by further washing the TRF-MIP@GNR nanocomposites with deionized water. For comparison, the NIP@GNR was prepared using the same synthetic approach in the absence of transferrin template. The synthesis of human serum albumin (HSA) imprinted gold nanorods followed the same procedure as the one designed for TRF-MIP@GNR. 2.6. SERS detection Aggregation of gold nanorods with the target protein. Gold nanorods with an amount of 5.9 mg were incubated with 1 mL
3. Results and discussion 3.1. Design of molecularly imprinted plasmonic nanosensors Gold nanorods exhibit transverse and longitudinal surface plasmon resonances that are deemed to be more SERS-active and to enable stronger Raman enhancement compared with spherical gold nanoparticles (Ni et al., 2008; Nikoobakht and El-Sayed, 2003). In this study, the gold nanorods (GNR) were prepared using the seed-mediated method together with anisotropic oxidation according to previous reports (Ming et al., 2008; Ni et al., 2008). Transmission electron microscopy (TEM) image shown in Fig. 1a confirms the uniform size of GNR with an average diameter of 267 2 nm and length of 8075 nm representing an aspect ratio (the length to width ratio) of 3.1 70.4 nm. The UV-vis spectrum of GNR dispersion is shown in Fig. 1b. The longitudinal surface plasmon resonance (LSPR) peak centered at 511 nm corresponds to the initial gold nanoparticle seeds. A new peak with stronger intensity appears at 775 nm and proves the growth of gold nanorods. Transferrin (TRF, pI 5.2-6.2; molecular weight 77.0 kDa), which was used as the template molecule, is iron-binding blood plasma glycoprotein. It is regarded as a protein biomarker that controls the level of free iron in biological fluids (Crichton and CharloteauxWauters, 1987). This protein was first adsorbed at the surface of the GNR via electrostatic interactions, and then the polydopamine (PDA) thin layer was prepared at the surface of GNR with attached transferrins via the self-polymerization of dopamine. The embedded protein templates were extracted with 10% (w/v) sodium dodecyl sulphate solution for 5 min to produce the molecularly imprinted layer. The polymer layer at the MIP@GNR core-shell structure had a thickness of 5.3 70.4 nm (Fig. 1c). A thin layer is favorable since it facilitates rapid mass transfer during protein adsorption and desorption. For comparison, non-imprinted
436
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
Fig. 1. TEM image of gold nanorods (a), UV-vis spectrum of gold nanorods dispersion (b), TEM image of transferrin-imprinted gold nanorods (c), FTIR spectra of nonimprinted (black line) and transferrin-imprinted gold nanorods (red line) (d), and XRD patterns of gold nanorods (red line), their counterparts coated with a thin layer of polydopamine (blue line), and the simulated pattern (bottom) (e).
polymer (NIP) thin layer was also prepared on the surface of gold nanorods using the same conditions except for the absence of protein templates. Energy-dispersive X-ray spectroscopy revealed 8.9 wt% nitrogen for MIP@GNR which demonstrated the successful coating of GNR with polydopamine layer. FTIR spectra of MIP@GNR and NIP@GNR nanocomposites shown in Fig. 1d exhibit peaks centered at 3430, 3145, 1636, and 1400 cm-1 ascribed to the stretching vibrations of hydroxyl, amine, C-N, and phenyl groups of polydopamine layer. Further characterizations of GNR and MIP@GNR were achieved using X-ray diffraction (XRD) patterns (Fig. 1e) that feature diffraction peaks typical of gold nanorods according to the JCPDS card (No. 04-0784). 3.2. Rebinding performances of protein-imprinted gold nanorods Both TRF-imprinted and non-imprinted gold nanorods were incubated in a transferrin solution. Their adsorption kinetic curves were presented in Fig. 2a. While the NIP@GNR binding capacity for transferrin was 16.7 mg/g, 66.6 mg/g of transferrin was found for MIP@GNR. The adsorption of transferrin on NIP@GNR originated merely from the non-specific binding. In contrast, imprinting significantly enlarged the adsorption capacity of MIP@GNR and resulted in an imprinting factor of 4.0. Thanks to the small thickness of the polymer layers, the equilibrium adsorption was achieved in a remarkably short time of only 7 min for NIP@GNR and 15 min for MIP@GNR. The slightly faster binding kinetics on NIP can be attributed to the easy diffusion to and fast interaction of transferrin with the non-specific sites located at the polymer surface. The adsorption capacity and adsorption/desorption kinetics of our imprinted nanocomposites are comparable or even exceed those
observed by other authors for the core-shell MIP nanosystems (Gao et al., 2011; Li et al., 2015d; Wan et al., 2015; Zhang et al., 2010; Zhang et al., 2012). The rebinding specificity and selectivity of the TRF-imprinted GNR was evaluated using adsorption capacities for the target protein and potentially interfering blood plasma proteins including myoglobin (pI 7.07; molecular weight 17.5 kDa), lysozyme (pI 11.0; molecular weight 14.3 kDa), and human serum albumin (pI 4.64; molecular weight 66.0 kDa). Fig. 2b confirms similar affinity for all four proteins on the non-imprinted surface. In contrast, the TRF-imprinted surface exhibited excellent selectivity towards its own template protein transferrin compared to a significantly lower affinity for other proteins. The imprinting factors (IF) of TRF-imprinted surface for myoglobin, lysozyme, and human serum albumin were only 1.2, 1.4, and 1.2, respectively. To investigate the general feasibility of our imprinting approach, human serum albumin was also used as the template molecule to prepare HSA-imprinted PDA@GNR nanosensor via the same synthetic procedure of self-polymerization. Similar to the transferrin imprinted system, the HSA-MIP@GNR exhibited a large adsorption capacity of 92.4 mg/g for human serum albumin that compared favorably to the value of 23.7 mg/g observed on the NIP (Fig. 2c). Binding experiments using other proteins on both MIP and NIP confirmed the high selectivity and specificity of the HSAimprinted polymer layer, which has imprinting factors of 3.9, 1.1, 1.9, and 1.5 for the template protein, myoglobin, lysozyme, and transferrin, respectively.
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
437
Fig. 2. The rebinding curves of the TRF-imprinted and non-imprinted polydopamine layers towards transferrin (a), the adsorption capacities of the TRF-imprinted, and nonimprinted polydopamine layers towards transferrin (TRF), myoglobin (MYO), lysozyme (LYZ), and human serum albumin (HSA) (b), and the adsorption capacities of the HSAimprinted, and non-imprinted polydopamine layers towards human serum albumin (HSA), myoglobin (MYO), lysozyme (LYZ), and transferrin (TRF) (c). (Protein concentration, 2.86 mg/mL).
3.3. SERS performances To study the performances of the TRF-imprinted nanosensors in SERS, both MIP@GNR and NIP@GNR were first incubated with transferrin solutions having a concentration of 2.0 10-6 mol/L to reach their adsorption equilibrium. In this way, the imprinted cavities of MIP@GNR were filled with transferrin. Then 2.5 mol/L of NaCl solution with pH value adjusted to 10 was added to initiate the aggregation of the nanorods and formation of “hot spots” (Scheme 1a). Raman spectra of TRF@MIP@GNR and TRF@NIP@GNR aggregates presented in Fig. 3a show that spectrum of the imprinted nanosensor incubated in transferrin is significantly enhanced. The characteristic peaks can be assigned to the vibrations of amide I (1626 cm-1) and amide III (1221 cm1 ) of α-helix structure, C-O and C-N stretching vibrations (1077 cm-1), C-C vibrations (952 cm-1), COO- vibrations (1399 cm-1), Cys (457 cm-1), Met (726 cm-1), Ala (892, 952 cm-1), and aromatic residues of Trp (590, 1531 cm-1), and Tyr (843 cm-1) of transferrin. In contrast, the enhancements in SERS spectra acquired for transferrin applied to both aggregated NIP@GNR and gold nanorod colloids were very weak. In all spectra, the GNR background is characterized with peaks centered at 761 and 1448 cm-1. The large difference in signal strength clearly results from the presence of specific binding sites contained in the imprinted polymer shell that selectively adsorb the protein. In addition to the above experiments using TRF@MIP@GNR aggregates, the SERS determination of transferrin was also carried out with samples prepared using simple application of the transferrin bioconjugate on a glass plate (Scheme 1b) as well as using the procedure reported by Cecchini et al. (Cecchini et al., 2013) that leads to self-assembled TRF@MIP@GNR in close-packed arrays at the liquid/liquid interface (Scheme 1c). The spectra are shown in Fig. 3b. The SERS spectrum acquired from sample at the glass plate
exhibited strong signals although with a reduced number of prominent peaks observed for C-C and C-N stretching vibrations (952 and 1123 cm-1), COO- vibrations (1362 cm-1), and Phe (1576 cm-1). The close-packed arrays featured more characteristic peaks attributed to Cys (469 cm-1), amide I (1629 cm-1) and amide III (1206 cm-1), C-C and C-N stretching vibrations (956, 1063 cm-1), Phe (1562 cm-1), Trp (1504 cm-1), and Tyr (657, 835 cm-1) but with significantly weaker intensities compared to those observed for aggregated TRF@MIP@GNR conjugate. The TRF-imprinted polydopamine gold nanorods were also incubated in transferrin solutions with low concentrations in order to elucidate the limits of sensitivity enabled by the MIP shell. Fig. 3c illustrates the Raman spectra of transferrin monitored in a concentration range of 5.6 10-8 mol/L to 2.1 10-6 mol/L confirming the appearance of identifiable Raman peaks even at a concentration as low as 5.6 10-8 mol/L. The insert in the Figure demonstrates a linear relationship between the integrated intensity of the peaks centered at 1395 cm-1 and the concentration of transferrin. These results also indicate that the detection limit of our imprinted plasmonic nanosensors is 10-8 mol/L. Identically, to demonstrate the versatility of our imprinting/SERS method, the HSA-imprinted polydopamine gold nanorods were incubated with 2.6 10-6 mol/L human serum albumin, the aggregated HSA@PDA@GNR bioconjugates were applied for the SERS detection. The SERS spectrum of human serum albumin presented in Fig. 4 (a) shows vibrations of amide I (1637 cm-1) and amide III (1250 cm-1) bands due to the α-helix structure, C-N stretching vibrations (1086 cm-1), COO- vibrations (1389 cm-1), Met (719 cm-1), Val (925 cm-1), Ala (951 cm-1), and aromatic residues such as Phe (1025 and 1580 cm-1), Tyr (669 and 841 cm-1), and Trp (561, 753, 1180, and 1543 cm-1) that are typical of human serum albumin.
438
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
Fig. 3. a) SERS spectra of gold nanorods (GNRs), TRF@GNR aggregates, TRF@NIP@GNR aggregates, and TRF@MIP@GNR aggregates (transferrin concentration, 2.0 10-6 mol/ L); b) SERS spectra of protein imprinted gold nanorods stacked onto a glass plate, and self-assembly into close-packed arrays after incubation with 2.0 10-6 mol/L transferrin; c) SERS spectra of transferrin determined on TFR@MIP@GNR aggregates with different concentrations ranging from 2.1 10-6 mol/L to 5.6 10-8 mol/L; d) SERS spectra of TFR@MIP@GNR aggregates with different thickness of polydopamine layer prepared from dopamine monomer solutions with concentrations ranging from 0.5 to 4.0 mg/mL (transferrin concentration, 2.0 10-6 mol/L); and e) SERS spectra of TFR@MIP@GNR aggregates prepared from nanorods with different aspect ratios (transferrin concentration, 2.0 10-6 mol/L).
3.4. Effects of the thickness of molecularly imprinted polymer layer on SERS detection The thickness of molecularly imprinted polymer shell is important for the function of the sensor. It is known that the thickness of the MIP shell needs to be preferably less than one particle
radius to guarantee that the target molecule is in the close vicinity of gold nanorods and to achieve the largest enhancement of the SERS signal (Bompart et al., 2010). A thinner layer facilitates mass transfer to and from the imprinted sites. However, the number of the available sites is low and the overall loading capacity is limited. Also, the imprinted cavities in a too thin layer may not have the
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
439
Fig. 4. a) SERS spectra of HSA-imprinted gold nanorods bioconjugates after incubation with 2.6 10-6 mol/L human serum albumin, and b) SERS spectra of TRF-imprinted gold nanorods bioconjugates after incubation with 2.0 10-6 mol/L transferrin in human serum.
perfect shape. Opposite applies to the thick layer. With the increase of layer thickness, the distance between the aggregated GNR is increased, and the probability of formation of “hot spots” that enhance the signal will decrease rapidly. Therefore, an optimum layer thickness must be found. The thickness of the layer is controlled by the concentration of dopamine in the monomer solution. In our experiments, we used concentrations ranging from 0.5 to 4.0 mg/mL. As expected, a high dopamine concentration of 4.0 mg/mL generates a 8.7 70.5 nm thick polydopamine layer on the surface of gold nanorods as estimated from TEM images shown in Fig. S1 in the Supporting Information. In contrast, Fig. 1c demonstrates that the polymer layer thickness was 5.3 70.4 nm while using 2.0 mg/mL dopamine. When the concentration of dopamine further decreased to 1.0 and 0.5 mg/mL, the polymer layer thickness on the surface of gold nanorods was 3.5 70.3 and 1.270.3 nm, respectively (Fig. S1). Our MIP@GNR bioconjugates differing in thickness of polydopamine layer and incubated with 2.0 10-6 mol/L transferrin solution were aggregated, and then used for the SERS detection. Fig. 3d illustrates that the nanosensor prepared using 2.0 mg/mL dopamine solution provided the most enhanced Raman scattering intensity. 3.5. Effect of aspect ratio of gold nanorods The use of gold nanorods with different aspect ratios enables tuning of the surface plasmon band to the wavelength of excitation laser to obtain the largest enhancement. Therefore, effects of aspect ratio of GNR on Raman enhancement were also studied and a series of gold nanorods with different aspect ratios were prepared. The transmission electron microscopy images shown in Fig. 5 confirm the successful preparation of nanorods with aspect ratio of 4.3 7 0.4, 3.4 70.3, and 2.9 70.3, respectively. The TRF@MIP@GNR aggregates were then subjected to Raman scattering measurements. Fig. 3(e) illustrates that the TFR@MIP@GNR aggregates prepared from nanorods with an aspect ratio of 2.9 70.3 exhibited the strongest Raman enhancement. 3.6. Practical application using real-life sample We also tested the performance of our imprinted plasmonic nanosensors in the detection of protein biomarker in real biological samples. The TRF-imprinted PDA@GNR was incubated with human serum spiked with 2 10-6 mol/L transferrin. After incubation, the nanocomposites were collected by centrifugation,
washed with Tris-HCl buffer, and the bound transferrin was detected by Raman spectroscopy. The Raman spectrum in Fig. 4b shows prominent peaks centered at 453 cm-1, 729 cm-1, 835 cm-1, 960 cm-1, 1060 cm-1, 1202 cm-1, 1395 cm-1, and 1536 cm-1 that are attributed to transferrin. Besides, we also observed some very weak characteristic bands at 567 cm-1, 673 cm-1, and 1270 cm-1 which might be ascribed to human serum albumin due to the nonspecific adsorption. A relative standard deviation of 8.4% was calculated from measurements of three parallel human sera, and the recovery was 98.6%. This result confirmed that our imprinted nanosensor has both high selectivity and specificity for the trace concentration of target protein even if used with real biological samples, and indicated a great potential and practical applicability of our approach in the detection of protein biomarkers applicable for clinical diagnostics.
4. Conclusions We have presented the rational design of new plasmonic imprinted nanosensors, which were applied for the selective “fishing-out” and sensitive SERS detection of the target protein from both aqueous solutions and human serum samples. Competitive rebinding studies with potentially interfering proteins and real life serum sample demonstrated that the imprinted polymer layer had a high selectivity for the targeted protein biomarker. Formation of the TRF@MIP@GNR aggregates enabled SERS detection of the protein presented at a concentration of at least 10-8 mol/L. Our system appears to be a promising tool for the early diagnosis and prognosis of diseases that are manifested with protein biomarkers.
Acknowledgment The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (21576017, 21436002, and 21306006), 973 programs (2014CB745103, 2013CB733603), 863 programs (2015BAD15B07), the Xiamen Scientific and Technological project (3502Z20142012), the Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology, and the Fundamental Research Funds for the Central Universities (YS1407, buctrc201413).
440
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
Fig. 5. TEM images of gold nanorods with different aspect ratios of 4.3 7 0.4 (a, b), 3.4 70.4 (c, d), and 2.9 7 0.3 (e, f).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.01.092.
References Ben-Amram, Y., Tel-Vered, R., Riskin, M., Wang, Z.G., Willner, I., 2012. Chem. Sci. 3, 162–167. Bompart, M., De Wilde, Y., Haupt, K., 2010. Adv. Mater. 22, 2343–2348.
Cai, D., Ren, L., Zhao, H., Xu, C., Zhang, L., Yu, Y., Wang, H., Lan, Y., Roberts, M.F., Chuang, J.H., Naughton, M.J., Ren, Z., Chiles, T.C., 2010. Nat. Nanotechnol. 5, 597–601. Cecchini, M.P., Turek, V.A., Paget, J., Kornyshev, A.A., Edel, J.B., 2013. Nat. Mater. 12, 165–171. Chang, L., Ding, Y., Li, X., 2013. Biosens. Bioelectron. 50, 106–110. Chen, S., Li, X., Zhao, Y., Chang, L., Qi, J., 2014. Chem. Commun. 50, 14331–14333. Crichton, R.R., Charloteaux-Wauters, M., 1987. Eur. J. Biochem. 164, 485–506. Daniel, S., Matikainen, A., Turunen, J., Vahimaa, P., Nuutinen, T., 2015. J. Colloid Interface Sci. 437, 119–123. Feng, S., Gao, F., Chen, Z., Grant, E., Kitts, D.D., Wang, S., Lu, X., 2013. J. Agric. Food. Chem. 61, 10467–10475. G, W., A, S., 1972. Angew. Chem. Int. Ed. 11, 341–348.
Y. Lv et al. / Biosensors and Bioelectronics 80 (2016) 433–441
G, W., A, S., K, Z., 1973. Tetrahedron Lett. 44, 4329–4332. Gao, R., Kong, X., Wang, X., He, X., Chen, L., Zhang, Y., 2011. J. Mater. Chem. 21, 17863–17871. Hanash, S.M., Pitteri, S.J., Faca, V.M., 2008. Nature 452, 571–579. Holford, T.R.J., Davis, F., Higson, S.P.J., 2012. Biosens. Bioelectron. 34, 12–24. Kamra, T., Zhou, T., Montelius, L., Schnadt, J., Ye, L., 2015. Anal. Chem. 87, 5056–5061. Kantarovich, K., Tsarfati, I., Gheber, L.A., Haupt, K., Bar, I., 2009. Anal. Chem. 81, 5686–5690. Kantarovich, K., Tsarfati, I., Gheber, L.A., Haupt, K., Bar, I., 2010. Biosens. Bioelectron. 26, 809–814. Li, D.Y., Qin, Y.P., Li, H.Y., He, X.W., Li, W.Y., Zhang, Y.K., 2015a. Biosens. Bioelectron. 66, 224–230. Li, N., Qi, L., Shen, Y., Qiao, J., Chen, Y., 2014a. ACS Appl. Mater. Inter. 6, 17289–17295. Li, Q., Yang, K., Liang, Y., Jiang, B., Liu, J., Zhang, L., Liang, Z., Zhang, Y., 2014b. ACS Appl. Mater. Inter. 6, 21954–21960. Li, S., Yang, K., Liu, J., Jiang, B., Zhang, L., Zhang, Y., 2015b. Anal. Chem. 87, 4617–4620. Li, X., Zhang, B., Li, W., Lei, X., Fan, X., Tian, L., Zhang, H., Zhang, Q., 2014c. Biosens. Bioelectron. 51, 261–267. Li, X., Zhang, B., Tian, L., Li, W., Zhang, H., Zhang, Q., 2015c. Sens. Actuators, B 208, 559–568. Li, Y., Bin, Q., Lin, Z., Chen, Y., Yang, H., Cai, Z., Chen, G., 2015d. Chem. Commun 51, 202–205. Liu, Y., Fang, S., Zhai, J., Zhao, M., 2015. Nanoscale 7, 7162–7167. Lv, Y., Tan, T., Svec, F., 2013. Biotechnol. Adv. 31, 1172–1186. Ma, Y., Xu, S., Wang, S., Wang, L., 2015. TrAC, Trends Anal. Chem. 67, 209–216. Ming, T., Kou, X., Chen, H., Wang, T., Tam, H.L., Cheah, K.W., Chen, J.Y., Wang, J., 2008. Angew. Chem. Int. Ed. 47, 9685–9690. Mok, J., Mindrinos, M.N., Davis, R.W., Mehdi, J., 2014. PNAS. 111, 2110–2115.
441
Ni, W., Kou, X., Yang, Z., Wang, J., 2008. ACS Nano. 2, 677–686. Nikoobakht, B., El-Sayed, M.A., 2003. Chem. Mater. 15, 1957–1962. Sykora, S., Cumbo, A., Belliot, G., Pothier, P., Arnal, C., Dudal, Y., Corvini, P.F.X., Shahgaldian, P., 2015. Chem. Commun. 51, 2256–2258. Tothill, I.E., 2009. Semin. Cell Dev. Biol. 20, 55–62. Vlatakis, G., Andersson, L.I., Muller, R., Mosbach, K., 1993. Nature 361, 645–647. Wackerlig, J., Lieberzeit, P.A., 2015. Sens. Actuators, B 207, 144–157. Wan, W., Han, Q., Zhang, X., Xie, Y., Sun, J., Ding, M., 2015. Chem. Commun. 51, 3541–3544. Whitcombe, M.J., Chianella, I., Larcombe, L., Piletsky, S.A., Noble, J., Porter, R., Horgan, A., 2011. Chem. Soc. Rev. 40, 1547–1571. Wu, Y.Y., Yang, C.X., Yan, X.P., 2015. Analyst. 140, 3107–3112. Xue, J.Q., Li, D.W., Qu, L.L., Long, Y.T., 2013. Anal. Chim. Acta 777, 57–62. Yang, J.R., Xie, S.M., Liu, H., Zhang, J.H., Yuan, L.M., 2015. Chromatographia 78, 557–564. Yang, K., Zhang, L., Liang, Z., Zhang, Y., 2012. Anal. Bioanal. Chem. 403, 2173–2183. Ye, J., Chen, Y., Liu, Z., 2014. Angew. Chem. Int. Ed. 53, 10386–10389. Zhang, M., Huang, J., Yu, P., Chen, X., 2010. Talanta 81, 162–166. Zhang, S.R., Li, J., Du, D.Y., Qin, J.S., Li, S.L., He, W.W., Su, Z.M., Lan, Y.Q., 2015a. J. Mater. Chem. A 3, 23426–23434. Zhang, W., He, X.W., Chen, Y., Li, W.Y., Zhang, Y.K., 2012. Biosens. Bioelectron. 31, 84–89. Zhang, Z., Guan, Y., Li, M., Zhao, A., Ren, J., Qu, X., 2015b. Chem. Sci. 6, 2822–2826. Zhao, X.L., Li, D.Y., He, X.W., Li, W.Y., Zhang, Y.K., 2014. J. Mater. Chem. B 2, 7575–7582.
