New Strategy for Label-Free and Time-Resolved Luminescent Assay

Jan 5, 2011 - Edison B. Gibelli , Jiang Kai , Ercules E.S. Teotonio , Oscar L. Malta , Maria C.F.C. Felinto , Hermi F. Brito. Journal of Photochemistr...
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New Strategy for Label-Free and Time-Resolved Luminescent Assay of Protein: Conjugate Eu3þ Complex and Aptamer-Wrapped Carbon Nanotubes Xiangyuan Ouyang,† Ruqin Yu,† Jianyu Jin,† Jishan Li,*,† Ronghua Yang,*,† Weihong Tan,† and Jingli Yuan‡ †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082, China ‡ State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116012, China

bS Supporting Information ABSTRACT: We report here a carbon nanotube-based approach for label-free and time-resolved luminescent assay of lysozyme (LYS) by engineering an antilysozyme aptamer and luminescent europium(III) (Eu3þ) complex. The sensing mechanism of the approach is based on the exceptional quenching capability of carbon nanotubes for the proximate luminescent Eu3þ complex and different propensities of single-stranded DNA and the DNA/protein complex to adsorb on carbon nanotubes. The luminescence of a mixture of chlorosulfonylated tetradentate β-diketone-Eu3þ and the antilysozyme aptamer was efficiently quenched by single-walled carbon nanotubes (SWNTs) unless the aptamer interacted with LYS. Due to the highly specific recognition ability of the aptamer for the target and the powerful quenching property of SWNTs for luminescence regents, this proposed approach has a good selectivity and high sensitivity for LYS. In the optimum conditions described, >700-fold signal enhancement was achieved for micromolar LYS, and a limit of detection as low as 0.9 nM was obtained, which is about 60-fold lower than those of commonly used fluorescent aptamer sensors. Moreover, due to the much longer lifetime of the Eu3þ luminescence than those of the ubiquitous endogenous fluorescent components, the timeresolved luminescence technique could be conveniently used for application in complicated biological samples. LYS concentrations in human urine were thus detected using time-resolved luminescence measurement with satisfactory recoveries of 95-98%.

probes for monitoring protein and nucleic acids in cell media.14,15 Nevertheless, the fluorescence lifetime of the pyrene excimer (∼60 ns) is not long enough, causing its measurement to entail complicated instrumentation and data interpretation. Therefore, continuous development of longer lifetime luminescence technologies for protein assay is still under way. As one of the best families of metal luminescent complexes, lanthanide ion compounds show highly desirable spectral characteristics, including relatively high quantum yields, large Stokes shifts (200-300 nm), narrow emission bands, and long luminescence lifetimes (∼1.0 ms) under ambient conditions.16 Especially, the long luminescence emission lifetimes allow lanthanide ion compounds to be used in time-resolved luminescence detection, reducing the background interference from ubiquitous endogenous fluorescent components.17-22 For example, lanthanide ion compounds were successfully applied in enzyme-linked immunosorbent assay (ELISA)23,24 and DNA-based sensors25,26 by covalently labeling the lanthanide ion chelates on the proteins or nucleic acid molecules. Though the very low background signal greatly improved the signal-to-background ratio (S/B), the

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he detection and quantification of proteins in complicated biological fluids play a pivotal role in academic research or medical diagnosis.1-3 On the basis of the difference of molecular recognition elements, protein assay can be divided into immunology techniques and synthetic nucleic acid ligand (aptamer) biosensing.4,5 Compared with antibody-antigen reaction-based immunoassay, aptamer-based biosensors (aptasensors) have advantages of easy preparation, stability, reusability, and the general availability for almost any given protein.6,7 Key factors, including a highly selective molecular recognition element and a novel signal transduction mechanism, have to be engineered together for successful development of an aptasensor. Generally, for an aptasensing system, target recognition can be transduced into electrical or optical readout.8,9 Among these signal transduction protocols, fluorescence detections have received much attention, because they are the most sensitive with several types of physicochemical interactions that can be used to modulate the label emission within the reaction complex.10-12 However, owing to diffusion and natural fluorescence of various compounds or proteins in biological samples,13 conventional fluorescent dyes suffer from serious limitations of sensitivity when used with complicated biological samples due to the high background signal from natural fluorescent compounds. Recently, Tan et al. have engineered excimer lifetime-based nucleic acid r 2011 American Chemical Society

Received: August 27, 2010 Accepted: December 13, 2010 Published: January 5, 2011 782

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complicated and expensive labeling process limited their application as common biosensing approaches. Nanomaterials possess unique properties that are amenable to biosensor applications by combining the highly specific recognition ability of biomolecules with the unique structural character of the nanostructure.27-29 Due to their unique, structurally defined optical and electronic properties,30-32 carbon nanotubes (CNTs) have become promising nanomaterials for application as biological transporters and selective cancer cell destructors,33 nanovehicles for drug delivery,34 and biosensors of DNA and proteins.35,36 Recently, it has been proved that organic dyes can be adsorbed on the surface of CNTs and that this produces fluorescence quenching.37-39 Also, single-stranded (ss) DNA has been demonstrated to interact noncovalently with single-walled carbon nanotubes (SWNTs) by means of aromatic interactions between nucleotide bases and SWNT sidewalls.38 Interestingly, however, when the ssDNA interacts with its target, it does not possess this property.39 On the basis of this feature of SWNTs, a series of SWNT-based fluorescence sensing approaches have been recently developed by our group and others for the detection of DNA,40 protein,41 and metal ion.42,43 Considering the powerful quenching capability of SWNTs for organic dyes, we reasoned that the SWNTs should be used for lanthanide ion compounds as efficient nanoquenchers for biosensors. To demonstrate the feasibility of this hypothesis, we report herein our first attempt to realize label-free and time-resolved luminescence assay of lysozyme (LYS) by employing conjugation of a Eu3þ complex and oligonucleotides/SWNTs. LYS is a ubiquitous protein in cells, and the LYS level in serum and urine can be used as the clinical index for many diseases such as cancer, HIV, myeloid leukemia,44-47 etc. For this purpose, many methods and techniques have been developed for the rapid, accurate, and sensitive analysis of LYS.48-50 Although these approaches are specific and sensitive for LYS detection in buffer, they may lose their sensitivity in complex biological fluids due to the high background of ubiquitous endogenous fluorescent components. By using the Eu3þ complex and SWNTs as the signal transducer and time-resolved luminescence assay, highly sensitive and selective detection of LYS in human urine was realized in the present work.

UV-vis absorption spectra were measured on a Hitachi U-4100 spectrophotometer (Kyoto, Japan). The luminescence emission spectra of the BHHT/Eu3þ compound were obtained on a Perkin-Elmer LS 50B luminescence spectrometer. For timeresolved luminescence detection, the parameter settings were as follows: excitation wavelength, 335 nm; delay time, 50 μs; gate time, 1.0 ms; cycle time, 20 ms; excitation slit, 10 nm; emission slit, 10 nm. The emission intensity at 612 nm was then monitored, which has the best sensitivity. Preparation of Aptamer-Wrapped SWNT Conjugates. The carboxyl-functionalized SWNTs were sonicated in doubly deionized water for 5 h to give a homogeneous black solution. After the pretreatment, enough SWNTs were introduced into 500 μL of the Tris-HCl buffer containing 500 nM P, and the mixture was incubated for 2 h at room temperature. Then the centrifugation step was carried out at 3000 rpm to get rid of the SWNTs without DNA coating. The obtained supernatant (P/ SWNTs) was stored in a refrigerator at 4 °C before further usage. X-ray Photoelectron Spectroscopy (XPS). For XPS measurements, the samples of SWNTs, SWNTs/P, SWNTs/P/LYS, were subjected to centrifugation at a speed of 80 000 rpm for 1 h. After removal of the supernatants, the precipitates were collected and washed with ethanol three times and then dried in air at 50 °C. The materials were loaded into the sample chamber on double-sided copper tape, and care was taken to ensure that the sample particles completely covered the tape. XPS measurements were carried out on an Axis Ultra imaging photoelectron spectrometer (Kratos Analytical Ltd., United Kingdom). Monochromatic Al KR X-rays (1486.7 eV) were employed. The X-ray source was a 2 mm nominal X-ray spot size operating at 15 kV and 15 mA for both survey and high-resolution spectra. Survey spectra, from 0 to 1100 eV binding energy (BE), were recorded at a 160 eV pass energy with an energy step of 1.0 eV and a dwell time of 200 ms. High-resolution spectra were recorded at a 40 eV pass energy with an energy step of 0.1 eV and a dwell time of 500 ms, with a typical average of 12 scans. The operating pressure of the spectrometer was typically ∼10-8 mbar. The XPS spectrum was analyzed using Casa XPS software, and all spectra were calibrated to the binding energy of C 1s photoelectrons at 285.2 eV. LYS Detection. To assess the luminescence responses of the conjugates to LYS, different concentrations of LYS were added to the P/SWNT solution, and the control solution without target was obtained by addition of the same volume of water to the nanotube complex solution. The solutions obtained were incubated at room temperature for 30 min and then centrifuged at 3000 rpm for 5 min. The pellet comprising aggregates and bundles of nanotubes at the bottom of the centrifuge tube was discarded, and 300 μL of supernatant was collected. The obtained solution was then transferred into a 300 μL cuvette with the addition of a certain BHHT/Eu3þ compound before the luminescence measurement. For performance of LYS measurement in cell media, different amounts of LYS were dissolved in cell media and added into the P/SWNT solution. The subsequent steps were the same as described above. Real Sample Analysis. To apply the time-resolved luminescence measurement, LYS in human urine was detected. The urine sample was voluntarily provided by healthy people and stored at 4 °C. Before luminescence detection, the samples were 10-fold diluted with the buffer solution and spiked with 0, 100, 225, and 650 nM LYS, respectively.

’ EXPERIMENTAL SECTION Chemicals and Instruments. The antilysozyme aptamer (P, 50 -ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-30 )51 was synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China). It was dissolved in doubly deionized water (18.3 MΩ 3 cm) produced by a Millpore water purification system. LYS, bovine serum albumin (BSA), myoglobin (MYO), cytochrome c from horse heart (CYT C), hemoglobin human from bovine (HEM), and human thrombin (THR) used in the experiments were all purchased from Sigma and used as supplied. To ensure CNTs were well soluble in water to control the quantitative analysis, carboxyl-functionalized SWNTs, purchased from Carbon Nanotechnologies, Inc., Houston, TX., were used. All other reagents were of analytical reagent grade and were purchased from Fluka (Switzerland). EuCl3 solution was prepared by dissolving Eu2O3 in 0.01 M HCl(aq) and diluted with water. 4,40 Bis(100 ,100 ,100 , 200 , 200 ,300 ,300 - heptafluoro-400 ,600 -dioxohexan-600 -yl)o-terphenyl (BHHT) was synthesized by using a previous method.52 The stock solution of this organic ligand was prepared in ethanol. The binding buffer was 20 mM Tris-HCl (pH 7.4, 100 mM NaCl, 2 mM MgCl2). 783

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Figure 1. Schematic description of label-free luminescence assay of a protein based on aptamer-wrapped SWNTs and a Eu3þ complex: (A) BHHT/ Eu3þ-grafted SWNT and the photoinduced electron transfer, (B) LYS-induced fluorescence enhancement of aptamer-grafted SWNTs and the Eu3þ complex. In the absence of LYS, the SWNTs were wrapped by the ssDNA, so that they were well dispersed and remained in the supernatant, providing the quenching substrate for the Eu3þ chelates. While in the presence of LYS, interaction of the aptamer with LYS made it unable to disperse the SWNTs, after centrifugation to separate the SWNTs, BHHT/Eu3þ in solution emitted a strong luminescence.

’ RESULTS AND DISCUSSION

sol-gel image for the supernatant of aptamer-wrapped SWNTs (lane b), while for the supernatant of P/SWNTs with addition of LYS, there is a clear aptamer band on the gel image (lane c), indicating that the formed aptamer-target complex fell off the SWNT surface and stayed in the supernatant after centrifugation. Another gel electrophoresis measurement corresponding to Figure 2A was carried out to further verify the observed phenomenon. It can be seen from Figure 2B that the individual aptamer-wrapped SWNTs appeared as a sharp, fast-moving band (lane b). The appearance of continuous smears of lane c indicated that the P/LYS complex could not wrap along the sidewall of SWNTs as well as that without LYS, and the SWNTs were not stable in a salt medium, resulting in partial aggregation of SWNTs. Furthermore, the naked SWNTs could not penetrate into the gel at all (lane a). These results were consistent with those of previous literature,55 indicating that the P/LYS complex can influence the dispersion of SWNTs. More information for interactions among SWNTs, P, and LYS can be obtained by XPS. The samples of SWNTs, SWNTs/P, and SWNTs/P/LYS interacted via the method mentioned above. All precipitates were collected and dried to perform XPS. Although the three samples contain mainly elemental C, O, and N, as well as Na and Cl (the LYS measurement was performed in Tris-HCl buffer containing 100 mM NaCl), the emergence of two phosphorus peaks (P2s and P2p) (Figure 3,

Sensing Scheme. The sensing scheme of the approach is based on the noncovalent assembly of SWNTs and singlestranded DNA (ssDNA) and the ability of the SWNT complex thus formed to both effectively quench the fluorophore and, in the presence of a target, restore the luminescence signal compared to that without a target. Figure 1 illustrates the signaling mechanism of the proposed approach for LYS assay. In aqueous solution, the SWNTs exist as aggregates, and binding of the P (antilysozyme aptamer) with the SWNTs disperses the aggregates that are individual entities in the solution, so that they adsorb the Eu3þ/BHHT complex, through π-π stacking interaction, and thus quench the Eu3þ ions luminescence emission. On the other hand, since the binding rate of P and the nanotube is lower than that of the DNA-protein interaction, competitive binding of the target with the SWNTs for the aptamer reduces the ability to disperse the nanotube’s aggregates so that there are no SWNTs in solution by a centrifugation step.53,54 Therefore, after addition of Eu3þ and BHHT to the aptamer solution, Eu3þ thus emitted strong luminescence emission compared with the luminescence of the P/SWNT assembled complex. The characteristic of the P/SWNT and LYS system was also investigated through sol-gel electrophoresis (Figure 2A). The results show that there is no significant antilysozyme aptamer band on the 784

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Figure 4. Luminescent emission spectra of BHHT (a, black), Eu3þ alone (b, red), BHHT/Eu3þ solution (c, blue), and a mixture of BHHT/ Eu3þ and SWNTs (d, green) in the Tris-HCl buffer. Inset: quenching efficiency (QE) of SWNTs for BHHT/Eu3þ as a function of the amount of SWNTs. The concentration of BHHT/Eu3þ is 500 nM, and λex = 335 nm. The peak assigned to 670 nm is scattered excitation light.

Figure 2. Electrophoretic analysis of the interaction of LYS and aptamer-wrapped SWNTs. (A) The reaction mixture containing P/ SWNTs was first incubated with (lane c) and without (lane b) 5.0 μM LYS. Then centrifugation at 80000 rpm was carried out, and 15 μL of the obtained supernatants was used for gel running. Key: lane a, 0.2 mg/mL SWNTs; lane d, 20 μM antilysozyme aptamer. Electrophoresis condition: 1% agarose gel, 100 V for 20 min. The gel image was obtained from Tanon 2500R. (B) Key: lane a, 0.2 mg/mL SWNTs; lane b, 0.2 mg/mL aptamer-wrapped SWNTs; lane c, 0.2 mg/mL aptamer-wrapped SWNTs þ 5.0 μM LYS. Electrophoresis condition: 1% agarose gel, 60 V for 30 min. The picture was taken by a normal camera. All the experiments were performed in 20 mM Tris-HCl (pH 7.4, 100 mM NaCl and 2 mM MgCl2).

Owing to the tetradenate structure and o-terphenyl chromophore, β-diketonate complexes are expected to possess the ability of metal center binding and pronounced absorption in the near-UV region for efficient energy transfer from the ligand to Eu3þ.16 Therefore, a tetradentate β-diketone derivative, 4,40 -bis(100 , 100 , 100 , 200 , 200 , 200 , 300 , 300 -heptafluoro-400 , 600 -dioxohexan-600 -yl)-o-terphenyl (BHHT; Figure 1), which appends two -C3F7 groups was selected as the Eu3þ ligand. The formation of the BHHT/Eu3þ complex was characterized by both UV-vis absorption spectroscopy and luminescence emission spectroscopy in Tris-HCl buffer solution. The maximum absorption peaks of the free ligand located at 334339 nm could be ascribed to the π f π* transition center on the o-terphenyl moiety. In the presence of Eu3þ, a red shift of maximum absorption wavelength was observed concomitant with a decrease of the absorption coefficient (Figure S1, Supporting Information). These results indicate that the tetradenate unit of the ligand is substantially coordinated to the center metal ion, which in turn perturbs the electronic cloud localized on the o-terphenyl ring. Figure 4 shows the luminescence emission spectra of BHHT/ Eu3þ. Room-temperature excitation of the ligand or EuCl3 solution, separately, did not lead to any measurable luminescence. With addition of BHHT to an aqueous solution of Eu3þ, appreciable Eu3þ characteristic emissions occurring at 596 and 612 nm (J = 1and 2), which indicate the 5D0 f 7Fj transition of Eu3þ,56 are observed concomitant with two side bands centered at 654 and 698 nm (J = 3 and 4). The remarkable enhancement of Eu3þ luminescence emission in the presence of BHHT suggests an interaction of the ligand with Eu3þ and, thus, the transfer of photonic energy absorbed by the ligand from the triplet excited state to the central Eu3þ. To obtain the stoichiometry and binding constant of Eu3þ with the ligands, the luminescence response of Eu3þ to different concentrations of BHHT was determined (Figure S2, Supporting Information). With detailed examination of the luminescence intensity changes of Eu3þ at 612 nm as a function of the ligand concentrations, the stoichiometry of BHHT with Eu3þ was determined to be 2:1 with an association constant of 6.39  1013 M-2 by using the curve fitting method reported previously.57 Next we explored the use of SWNTs as a quencher of BHHT/ Eu3þ. SWNTs have been proved superquenchers of fluorophores by way of energy-transfer and electron-transfer processes.40 On the other hand, the ligand BHHT is speculated to absorb onto the sidewall of SWNTs via π-π stacking,37 which quenched the

Figure 3. X-ray photoelectron spectra of SWNT (black curve), P/ SWNT (blue curve), and P/LYS/SWNT (red curve) precipitates deposited on glass electrodes. The inset shows considerable P peaks in the case of P/SWNTs.

blue curve) observed from SWNTs/P (phosphorus constitutes a fraction of DNA chemical composition but is not contained in LYS) indicates the aptamer was wrapped around the surface of the SWNTs. However, when P was first interacted with LYS and subsequently with SWNTs, one could not find a phosphorus peak in the XPS spectrum (Figure 3, red curve), suggesting that the aptamer/LYS complex did not adsorb on the SWNT surface but remained in the supernatant. Formation of a Eu3þ-Ligand Complex and Luminescence Quenching. Because direct excitation of a lanthanide ion is very inefficient, every lanthanide luminescent complex requires incorporation into a chromophore structure capable of transferring its excited-state energy to the encapsulated lanthanide ion. 785

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Figure 5. Steady-state emission spectra of P/SWNTs/BHHT/Eu3þ with increasing concentrations of LYS in the Tris-HCl buffer. The arrow indicates the signal changes with increasing LYS concentration. Inset: emission intensity enhancement (S/B) of P/SWNTs/BHHT/ Eu3þ at 612 nm as a function of the LYS concentration. λex = 335 nm.

Figure 6. Selectivity of P/SWNTs/BHHT/Eu3þ toward different proteins (0.1 μM, x-axis markers). Here I0 and I are the Eu3þ luminescence intensities of the system in the absence and the presence of protein, respectively. Excitation was at 335 nm, and emission was monitored at 612 nm.

luminescence by disturbing its incorporation with Eu3þ or transferring BHHT’s excited-state energy to SWNTs. The equation is present in Figure 1A. From the inset of Figure 4, we can see the luminescence intensity of the BHHT/Eu3þ solution was proportionally decreased with the addition of increasing amounts of SWNTs, indicating that more and more BHHT/Eu3þ complexes were adsorbed onto the surface of the SWNTs. When the concentration of SWNTs added reached about 0.05 mg/mL, the luminescence intensity of 500 nM BHHT/Eu3þ was nearly completely quenched (Figure S3, Supporting Information). Luminescence Sensing of LYS in Buffer. Because both the SWNTs and Eu3þ ligand used in the work could be protonated, the effects of pH on the luminescence feature and SWNT quenching ability were first studied. In the absence of SWNTs, a luminescence decrease of BHHT/Eu3þ was observed in the pH range of 7.0-5.0, because Hþ disrupted the combination of BHHT and Eu3þ through protonation of the ligand.58 There was almost no effect on the luminescence of BHHT/Eu3þ in a neutral or alkalescence medium (Figure S4, Supporting Information). In the presence of SWNTs, the luminescence of the Eu3þ complex could be effectively quenched by SWNTs at a pH range of 4.0-10.0 and Hþ had no significant interference in the interaction between BHHT/Eu3þ and SWNTs. To get a high S/B, as well as a physiologically compatible condition, pH 7.4 was selected for LYS sensing. The amounts of BHHT/Eu3þ affect the response behavior of the system for LYS. As shown in Figure S5 (Supporting Information), fixing the aptamer concentration, when the concentration of BHHT/Eu3þ was low, the luminescence of BHHT/Eu3þ, in the presence of LYS, was quenched by the remaining SWNTs, which resulted in a low luminescence signal and turned out a small S/B. However, when the concentration of BHHT/Eu3þ was too high, the luminescence could not be effectively quenched by SWNTs, leading to a high background signal and thus a low S/B. At a fixed aptamer concentration of 500 nM, 500 nM BHHT/Eu3þ was optimized to turn out a better S/B. With the optimized conditions, steady-state luminescence measurements were performed in Tris-HCl buffer solution. Figure 5 shows the luminescence emission spectra of P/SWNTs/BHHT/ Eu3þ with varying concentrations of LYS. The emission intensity of the 612 nm band was significantly increased with an increase in the LYS concentration. When the concentration of LYS was increased to 10.0 μM, the Eu3þ luminescence reached the maximum with a nearly

735-fold luminescence signal enhancement (Figure 5, inset). A detection limit as low as 0.9 nM was obtained, which is 60-fold lower than the value of 55.6 nM of the conventional assay,50 indicating that this approach has the ability of signal amplification. The reason is possibly that the reduction of a single SWNT resulted from one binding reaction event between the aptamer and target and could lead to many luminescent chelate molecules that stay free in the supernatant solution and give enhanced detection signals. To evaluate a special protein detection assay, selectivity is one of the most important factors, because it reflects the ability of the assay to avoid false-negative or false-positive readouts. To understand the response behaviors of the approach toward different targets, the Eu3þ luminescence intensity changes by different proteins (MYO, HEM, THR, BSA, CYT C, and LYS) were studied. As shown in Figure 6, when tested with MYO, HEM, and THR at a concentration of 0.1 μM, the system only gave a small Eu3þ luminescence response to these proteins compared with that without protein, while a slight luminescence decrease was observed when the system was tested with BSA or CYT C. However, significant Eu3þ luminescence enhancement occurred after the introduction of 0.1 μM LYS to the solution under the same conditions. This selectivity is similar to that of the original LYS aptamer, confirming that the conjugation of SWNTs with the aptamer does not affect the selectivity of the aptamer. However, it is worth noting that substrates that could interact with Eu3þ, such as a high concentration of multiphosphates and multicarboxylate ions, reduce the luminescence response of the system to LYS (Figure S6, Supporting Information). Nevertheless, the selective capability, along with its high sensitivity and simplicity, granted the assay great potential in clinical applications. LYS Assay in Cell Media. Assay of proteins in complex biological fluids, which contain ubiquitous endogenous components that produce a high fluorescence background,13 makes the commonly used biosensors fail to be efficient without sample pretreatment. To evaluate the luminescence characteristics of BHHT/Eu3þ in complex conditions, Figure 7A shows the steady-state emission spectra of BHHT/Eu3þ in Tris-HCl buffer solution and 1640 cell growth media. One can see from Figure 7A that the cell growth media had a high autofluorescence and dominated the luminescence spectra from 360 to 500 nm. Although the luminescence background at the BHHT/Eu3þ emission range was low, the S/B of the system for 1.0 μM LYS in cell media was lessened from 550 to 3.5 compared with that in buffer solution (Figure 7A, inset). These results suggest that 786

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Figure 7. Monitoring LYS in cell media: (A) steady-state emission spectra (λex = 335 nm) of cell media (black), P/SWNTs/BHHT/Eu3þ in the cell media (red), P/SWNTs/BHHT/Eu3þ þ LYS in the cell media (pink), P/SWNTs/BHHT/Eu3þ in the Tris-HCl buffer (green), and P/SWNTs/ BHHT/Eu3þ þ LYS in the buffer (blue). Inset: patterns of S/B for different reaction media resulting from the spectral detection at 612 nm. (B) Luminescence emission spectra of BHHT/Eu3þ in cell media with different delay time windows of 0 ms (blue) and 0.05 ms (red). (C) Time-resolved luminescence emission spectra of P/SWNTs/BHHT/Eu3þ in the cell growth media with increasing concentrations of LYS using a delay time of 0.05 ms and a gate time of 1.0 ms. The arrow indicates the signal changes with an increase in the LYS concentration. (D) Emission intensity enhancement of P/ SWNTs/BHHT/Eu3þ in cell media as a function of the LYS concentration using steady-state (a) and time-resolved (b) luminescence measurements. Here I0 and I are the Eu3þ luminescence intensities at 612 nm in the system in the absence and the presence of LYS, respectively. For comparison, all the luminescence emission spectra were normalized to the highest intensity.

LYS in the concentration range of 10 nM to 2.0 μM, and when the concentration of LYS reached 2.0 μM, the luminescence intensity gradually leveled off. A higher LYS concentration produced only a small intensity variation (Figure 7D). In Figure 7D, the value of I/I0 was estimated to be 56.2 in the presence of 2.0 μM LYS, while the value dropped to 4.3 upon the addition of 2.0 μM LYS using steadystate luminescence measurement. The variance by a factor of 56.2/ 4.3 = 13 was achieved, demonstrating the efficiency of the approach for direct quantification of LYS in complex biological fluids. It is worth noting that the dynamic detection range of the system for LYS is completely controlled by the concentration of the aptamer P. In our experiment the concentration of free aptamer could influence the amount of SWNTs dispersed in the supernatant, which provided the quenching substrate for the Eu3þ chelates. When the amount of aptamer was fixed, there was no aptamer to bind to excessive LYS to produce signal enhancement. Therefore, we could tune the sensing range by altering the concentration of the aptamer. Preliminary Application. The practical applicability of the present method was tested on the assay of LYS in human urine. The LYS level in urine can be used as the clinical index for myeloid leukemia and paunch, and it was reported that the urines of patients with renal diseases always contain LYS.46,47 Since human urine would contain biologically related metals, anions, or molecules, following the described procedure, the selectivity of the system for LYS in the presence of the interfering species in urine was investigated by adding different amounts of the interfering agent to the samples containing 1.0 μM LYS. The tolerance limit was taken

the approach is inefficient for protein assay in a complex environment. On the other hand, most of the background fluorescence of biological molecules and organic fluorophores has a lifetime of