Mass Amplifying Probe for Sensitive Fluorescence Anisotropy

Jun 11, 2012 - Gareth Jenkins,. ‡,§ and Chaoyong James Yang*. ,‡. ‡. Key Laboratory of Analytical Science, Key Laboratory for Chemical Biology ...
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Mass Amplifying Probe for Sensitive Fluorescence Anisotropy Detection of Small Molecules in Complex Biological Samples Liang Cui,‡ Yuan Zou,‡ Ninghang Lin,‡ Zhi Zhu,‡ Gareth Jenkins,‡,§ and Chaoyong James Yang*,‡ ‡

Key Laboratory of Analytical Science, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China § Institute of Biomedical Engineering, Imperial College London, South Kensington, London SW7 2AZ, U.K. ABSTRACT: Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.

F

information of the probe molecule upon target binding to correctly report the interaction.5,6 Second, FA is able to use only one dye labeling to report target molecules in real time and in homogeneous solutions without the need of a quencher or FRET pair, which simplifies the probe design and reduces synthesis cost. Third, FA is a ratiometric approach, thus it is insensitive to sample fluorescence fluctuation and photobleaching.7 Because of these advantages, FA is currently widely applied in diverse fields, especially in the life sciences, such as for biochemical research, drug discovery, clinical diagnosis, and food analysis.1,8−10 Recently, dye-labeled aptamers have also been successfully exploited for FA sensing of macromolecules. Compared to macromolecules such as proteins, aptamers as single-stranded oligonucleotides are relative small molecules. The binding of target proteins will yield a significant change in their molecular masses and therefore change in the rotational

luorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing.1 The FA value is sensitive to changes in the rotational motion of a fluorophore functionalized object, which in turn depends upon a number of parameters including molecular volume and molecular mass at a constant temperature and solution viscosity. That is, when a small fluorescent molecule is free in solution, it rotates at a rate commensurate with its size and the FA value is relatively small. If, however, the fluorescent molecule forms a complex with another molecule, its rotational rate decreases and the FA value increases, and the degree of variation depends on the strength of the binding interaction and the size of the complex.2,3 The relationship between the molecular mass of the fluorescent molecule and its FA value makes anisotropy an ideal method for the investigation of macromolecules and biomolecular interactions.4 Compared with other fluorescence signaling approaches, such as FRET or fluorescence quenching, FA signaling has several unique advantages. First, in theory, as long as the biological interaction induces a change in the rotation of the fluorescently labeled probe, FA should be applicable for the real-time analyzing of such an interaction. As a result, it does not require precise conformational change © 2012 American Chemical Society

Received: January 18, 2012 Accepted: June 11, 2012 Published: June 11, 2012 5535

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Table 1. Sequences of Probesa

diffusion rates of the labeling fluorophores, resulting in detectable changes in their FA values. Hieftje’s group first developed a FA method for the real-time detection of thrombin using aptamer.11 Since then the FA method has been further extended to the detection of other proteins, such as human neutrophil elastase,12 oncoprotein PDGF,13 IgE,14 angiogenin,15 and lysozyme.16 However, one of the important requirements for a workable FA probe is that the molecule being measured should induce a significant change in molecular mass before and after recognition.7,17 Thus, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins, and this approach is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. By using elegant design or the peculiar conformational flexibility of aptamers, only a few aptamer probes based on FA for small molecules have been reported.18−20 Unfortunately, these methods are quite limited by their complexity in design and could not be applied for all the small molecules. There are also some reports that used AuNPs for amplified FA detection of metal ions such as Hg2+, Cu2+, or Pb2+. However, these methods require complicated probe design and synthesis, and are only limited to metal ions.9,21 A general strategy in designing FA aptamer probes for small molecule analysis would be highly valuable to this field. In this work, inspired by allosteric regulation, which is the most common phenomenon in regulating the binding affinity or enzymatic activity of a protein through the binding of an effector molecule to the protein’s allosteric site and has been widely employed by many biochemists and bioanalysts,22−30 we have developed a molecular mass amplifying strategy to construct FA aptamer probes for small molecule analysis in complex biological samples. With the new method, we were able to use FA to directly detect ATP and cocaine with detection limits of 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were detected without any tedious sample pretreatment. Our results establish that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.

name

sequences

MAPATP blocking strand blocking strand blocking strand blocking strand blocking strand blocking strand MAPcocaine

3−6 4−6 5−6 6−6 7−6 8−6

cocaine-blocking strand

5′ - GGT TGG TGT GGT TGG ACC TGG GGG AGT ATT GCG GAG GAA GGT - FAM - 3′ 5′ - ACC TGG ACC - 3′ 5′ - A ACC TGG ACC - 3′ 5′ - CA ACC TGG ACC - 3′ 5′ - CCA ACC TGG ACC - 3′ 5′ - A CCA ACC TGG ACC- 3′ 5′ - CA CCA ACC TGG ACC- 3′ 5′ - FAM - AGAC AAG GAA AAT CCT TCA ATG AAG TGG GTC G GGT TGG TGT GGT TGG - 3′ 5′ - CAA CCC GAC CC - 3′

a

The italic letters represent thrombin aptamer sequence; the bold letters represent target aptamer sequence.

Afterward, the DNA product was spun at 14000 rpm at 4 °C for 10 min. The supernatant was removed, and the precipitated DNA product was dissolved in 500 μL of 0.1 M triethylamine acetate. HPLC purification was then performed with an Alltech C18 column on an Agilent 1100 series HPLC system (Agilent Technologies, Inc.). The purified DNA product was dried and detritylated via incubation with 200 μL of 80% acetic acid for 20 min. The detritylated DNA product was mixed with 400 μL of ethanol and dried using a vacuum dryer. The purified probe was dissolved in DNA grade water and quantified by determining the UV absorption at 260 nm on an Agilent 8453 UV/vis spectrometer, after which the probe was stored at −20 °C for future experiments. Fluorescence Measurements. FA signals were measured on a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon, Paris, France) using the L-format configuration. Excitation was set to 488 nm and emission was detected at 518 nm. All experiments were carried out at room temperature. In the ATP sensing experiment, the target solution was added to 100 μL of 20 mM Tris-HCl buffer solution (50 mM NaCl, 5 mM MgCl2, 5 mM KCl, pH 7.4) containing aptamer probe (100 nM) and blocking strand (150 nM), and the mixture was equilibrated at room temperature for 10 min prior to FA measurement. For cocaine, the parameters were the same as those for ATP except that the NaCl concentration of the buffer was 200 mM. Six anisotropy measurements were taken each time using an integration time of 0.1 s for each sample, and the resulting anisotropy values were averaged. Parameter Determination. Anisotropy (r) is a ratio, defined as the difference between linearly polarized components of emission divided by the total light intensity.3 The rotational diffusion of a fluorophore is a dominant cause of FA, and most applications depend on changes in the rate of rotation. According to the modified Perrin eq 1,3 the FA value of a rotating molecule is proportional to the viscosity of the solvent (η), size (v), ̅ and molecular mass (Mr) of the molecule

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EXPERIMENTAL SECTION Materials and Reagents. Thrombin was obtained from Haematologic Technologies, Inc. (Vermont, USA). All other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). The reagents for DNA synthesis were purchased from Proligo (Sigma-Aldrich Inc., St. Louis, MO), Glen Research (Sterling, VA, USA), and ChemGenes (Wilmington, MA, USA). DNA Synthesis. All oligonucleotides were synthesized inhouse on a Polygen 12-Column DNA/RNA synthesizer. Their sequences are listed in Table 1. The synthesis protocol was set up according to the requirements specified by the reagents’ manufacturers. Following on-machine synthesis, the DNA product was deprotected and cleaved from CPG by incubating with 0.5 mL of concentrated ammonium hydroxide for 8 h at 65 °C in a water bath. The cleaved DNA product was transferred to a 2 mL centrifuge tube and mixed with 50 μL of 3.0 M NaCl and 1.25 mL of ethanol, after which the sample was placed into a freezer at −20 °C for ethanol precipitation.

1 1 τRT 1 = + • r r0 r0η( v ̅ + h) Mr

(1)

where v ̅ is the specific volume of the molecule, and h is the hydration radius, T is the temperature in K, R is the molar gas constant (8.31 J/mol·K), and η is the viscosity in poise (P).3 5536

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Scheme 1. Working Principle of MAP for Sensitive Detection of Small Molecules Based on FAa

a

Double arrows represent the possible rotational contributions to the variation of the FA signal.

Figure 1. (A) The MAPATP performance with different sequences of the blocking strands. (B) Response of MAPATP to thrombin and ATP. 400 nM thrombin was added into a solution containing 100 nM MAPATP followed by 1 mM ATP.

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RESULTS AND DISCUSSION Working Principle of Mass Amplifying Probe (MAP). The design of a MAP is shown in Scheme 1. Overall, the probe is designed in such a way that only when the target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the complex. Specifically, the aptamer probe consists of a targeting aptamer domain (blue ribbon in Scheme 1) against the target molecule and molecular mass amplifying aptamer domain (purple ribbon) for binding with the amplifier protein. The probe is initially deactivated by a short blocking strand (cyan ribbon) which is partially complementary to both target aptamer and amplifier protein aptamer so that the molecular mass amplifying aptamer domain would not bind to the amplifier protein (Scheme 1a), unless the probe has been activated by the small molecule target (Scheme 1b). To demonstrate the concept, we first designed a MAP for ATP (MAPATP). The molecular mass of ATP is 507, and the aptamer against ATP is a 27 bp sequence with a molecular mass of 8486. It would be impossible to directly use the aptamer sequence to sense ATP using FA method because the binding of the ATP to its aptamer would only change 5% of the molecular mass, which is insignificant and would not produce an observable FA value change. The MAPATP consists of an

ATP aptamer domain with FAM labeling (5′- ACC TGG GGG AGT ATT GCG GAG GAA GGT -FAM- 3′) and mass amplifying aptamer domain for the amplifier protein. In this case, thrombin was chosen as the amplifier protein because of its high molecular mass (37000) and the availability of its aptamer (5′ - GGT TGG TGT GGT TGG - 3′). The 11 bp blocking strand (5′-CA ACC TGG ACC- 3′) binds to part of the ATP aptamer sequence and the thrombin aptamer sequence. The sequence of the blocking strand was designed to inhibit the binding of the thrombin aptamer to thrombin without the target molecule ATP, as the thrombin aptamer and blocking strand can form a very stable complex in buffer at room temperature. Under these conditions, the MAPATP remains free in solution, and the anisotropy value would not change with the addition of the amplifier protein thrombin. In the presence of ATP, however, ATP displaces part of the blocking strand through its binding to the ATP aptamer domain, leaving few base pairs between the thrombin aptamer and the blocking strand, which is unstable at room temperature and results in the release of the blocking strand from the aptamer strand. As a consequence, the thrombin aptamer domain is free to bind with thrombin, causing a dramatic change in the molecular mass of the MAPATP from 17129 to 50843, generating nearly a 300% mass change. From the modified Perrin eq 1, the FA value of a fluorophore depends on 5537

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Figure 2. (A) Effect of ATP concentrations on the FA of a solution containing MAPATP (100 nM) and thrombin (400 nM). FA value at low ATP concentrations. Measurements were made less than 2 min after mixing the solutions. Each point was the mean of six experiments with the standard deviation as the error bar. (B) FA response of MAPATP to CTP, GTP, UTP, and ATP. Experiments were performed in the presence of 100 nM MAPATP, 400 nM thrombin, and the target (CTP, GTP, UTP, and ATP) 1 mM.

rotational diffusion rate of the FAM-labeled aptamer strand. In this regard, the introduction of the thrombin as the mass amplifier into the solution provides FA signal amplification and improves the performance and sensitivity of the probe for detecting ATP in homogeneous solution. Meanwhile, it must be pointed out that the anisotropy change in this aptamer-ATP binding reaction occurs rapidly, within 1 min, which has the potential for the real time monitoring of ATP in homogeneous solution. Sensitive and Selective Detection of ATP. To test whether the FA change of the MAPATP can be used to quantitatively detect ATP, a titration experiment was carried out. Figure 2A plots the FA from solutions of 100 nM MAPATP with various ATP concentrations. The results showed that FA value increased as the concentration of the target increased. As shown in the insert of Figure 2A, a good linear relationship between the amount of ATP and the probe’s anisotropy signal was observed at the low ATP concentration (1−25 μM). The detection limit was found to be 0.5 μM, which is competitive with fluorescence-based aptasensors,32−35 but is slightly higher than electrochemical based methods.36,37 According to the modified Perrin eq 1, it is possible for further improvement in sensitivity by using an amplifying protein with higher molecular mass. In this way, the rotation of the fluorophore in the molecule would become much slower due to the higher molecular mass of the amplifier. In addition, the reaction was quick and reached equilibrium within 1 min. The rapid nature of the assay is of particular importance in field applications where individual samples must be analyzed in a short time. On the basis of the aptamer’s inherent affinity,38 the binding of probe and ATP was found to be highly selective. In order to demonstrate the selectivity of the method, we tested the MAPATP with molecules having structural similarities to ATP. A series of ATP analogues including GTP, CTP, and UTP were employed as controls, and no distinct fluorescent anisotropy changes were observed (Figure 2B), indicating an excellent selectivity of the MAPATP. Direct Detection of Target in Complex Biological Samples. The above results demonstrated that our MAP

the molecular mass of the rotating molecule. Thus, the anisotropy value of the solution would significantly increase. Probe Optimization. For the MAP, the length of the blocking strand is a critical factor. On one hand, if the blocking strand is too short, the probe should not be stable enough to prevent the thrombin aptamer domain from binding to thrombin when there is no ATP present. Thus, the background FA signal would be high. On the other hand, a longer blocking strand would inhibit the binding of ATP and result in a poor reporting signal. With an ideal length of the blocking strands, in the absence of target ATP, the MAPATP is stable enough to resist the binding with thrombin; while, in the presence of ATP, it can release the blocking strand and bind to both ATP and thrombin. Based on our rational analysis and Lu’s report,24,31 we first investigated the blocking strand that contains six bases complementary to ATP aptamer domain and various numbers of bases complementary to the thrombin aptamer domain (blocking strand 3−6, 4−6, 5−6, 6−6, 7−6, and 8−6; see Table 1 for the sequences). The probes were prepared by mixing 100 nM aptamer strand, 150 nM blocking strand, and 400 nM thrombin in the buffer. Anisotropy values were recorded both with and without the addition of 1 mM ATP to the solution. As shown in Figure 1A, in the absence (black line) or presence (red line) of ATP, the anisotropy value decreases with the number of blocking strand bases complementary to the thrombin aptamer domain increasing from 3 to 8. As exhibited in the insert of Figure 1A, when the length of blocking strand was 5−6, we obtained the maximal signal-background ratio (SBR). Thus, in this work, we chose a 5−6 bp blocking strand for MAPATP. The response of MAPATP to the addition of thrombin and ATP was monitored in real time. As shown in Figure 1B, with the addition of a high concentration of thrombin (400 nM) to the MAPATP solution, no apparent increase in FA was observed. This illustrated that the probes have formed a stable complex and did not respond to thrombin. By contrast, upon the addition of ATP (1 mM), a remarkable and sharp rise in FA was observed, indicating that the binding of ATP induced the conformation change of the probes to bind with thrombin, which resulted in increasing molecular mass and hindering the 5538

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Figure 3. (A) Time course study of MAPATP (100 nM) with different concentrations of ATP in cell media. (B) Effect of ATP concentrations on the FA of a solution containing a MAPATP (100 nM) and thrombin (400 nM) in cell media.

Figure 4. (A) Response of MAPcocaine (100 nM) to cocaine (black, 5 mM) and its metabolites (red, 5 mM). (B) Effect of cocaine concentration on the FA of a solution containing MAPcocaine (100 nM) and thrombin (400 nM). FA value change at low cocaine concentrations. Measurements were made less than 5 min after mixing the solutions. Each point was the mean of six experiments with the standard deviation as the error bar.

to be about 1 μM based on three times standard deviation of six measurements of blank samples. These results demonstrate the feasibility of direct quantitation of ATP in complex biological fluids. As reported, based on the same ATP aptamer, some FRET or fluorescence quenching methods can also detect ATP in complex biological samples such as cytosol buffer or cell directly, but the limits of detection were as high as 50−100 μM,39,40 which are 2 orders of magnitude less sensitive than our method. Generality of Mass Amplifying Strategy. Thus far, we have demonstrated that based on mass amplifying strategy with MAP, FA can be used for highly sensitive and selective detection of ATP in complex biological samples. By simply replacing the recognition region of MAPATP with other aptamers, other small targets should also be detected by the same strategy. To demonstrate the general applicability of the method, a MAP was designed for cocaine (MAPcocaine). The sequences of the probe are listed in Table 1. As shown in Figure 4A (black curve), the MAPcocaine was stable in solution. Even

works well in relative simple and pure buffer systems with excellent sensitivity and selectivity. However, the further application challenge of MAP is to tolerate any interference from complex biological samples. As mentioned above, one of the advantages of FA is being less affected by environmental interferences, such as background fluorescence.7 Thus, it can be used in complex biological samples. To demonstrate this advantage, cell media was employed as a model matrix for detection of ATP. Since the cell media contains a significant amount of autofluorescent species including proteins, riboflavin, nicotinamide, pyridoxine, and tryptophan, as well as porphyrin, the background fluorescence signal from these species spans the visible spectrum and masks the probe signal, making the homogeneous detection of biomolecules in cell media extremely difficult. In our experiment, the MAPATP was added to the cell media spiked with different concentrations of ATP (Figure 3). The FA intensity was found to increase proportionally with the increase of target ATP concentration in cell media. The limit of detection in the cell media was also found 5539

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Figure 5. (A) Time based study of MAPcocaine (100 nM) for different concentrations of cocaine in urine. (B) Effect of cocaine concentrations on FA of a solution containing MAPcocaine (100 nM) and thrombin (400 nM).

applicable. By simply replacing the aptamer domain with another aptamer sequence, the detection of different targets could be realized. Finally, because FA is less affected by environmental interferences, the target can be directly detected in complex biological samples without any tedious sample pretreatment. The unique properties of the mass amplifying strategy will enable the development of a new class of probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.

after addition of a high concentration of thrombin to the solution, there was still no apparent increase in FA signal. After the addition of 5 mM cocaine, a remarkable rise in FA was observed. As a control, upon addition of the same concentration of its metabolites (Figure 4A (red curve)), no apparent signal could be observed. The result indicates that we have successfully designed a new MAPcocaine. Figure 4B shows the response of MAPcocaine to different concentrations of cocaine in buffer. The linear range was from 1 to 100 μM, and the detection limit was found to be as low as 0.82 μM. The results were also comparable, or superior to many existing fluorescence-based aptasensors,41−43 indicating our design was a universal and sensitive method for small molecule analysis. A complex biological sample human urine, which was highly autofluorescence, was also used to test the feasibility of MAPcocaine for real sample analysis. Our data showed that as low as 5 μM of cocaine can be directly detected in urine without any tedious sample pretreatment (Figure 5). It has been reported that average cocaine concentration in urine is about 70 μM within 24 h after taking cocaine.44 The results suggest that our method has significant implications for real applications.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+)008605922187601. Fax: (+)008605922189959. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by National Basic Research Program of China (2010CB732402), National Scientific Foundation of China (21075104), and the Natural Science Foundation of Fujian Province for Distinguished Young Scholars (2010 J06004). L.C. and Y.Z. contributed equally to this work.

CONCLUSIONS

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In conclusion, we have developed a novel mass amplifying strategy to construct anisotropy aptamer probes MAP for small molecule detection, which contains the target aptamer domain, an amplifier-a thrombin aptamer domain, and a blocking strand that inhibits the binding of thrombin aptamer to thrombin without the target molecule. The mass amplifying strategy proposed here has several advantages. First of all, it makes it possible to use FA as a signal transduction mechanism to construct aptamer probes against small molecules. Taking MAPATP as an example, compared with normal aptamer design strategy with only 5% mass difference after target recognition which is difficult for FA detection, our MAPATP could cause at least 300% mass difference after target binding, resulting in an apparent FA value change for sensitive detection of small molecules. Second, because of the large molecular mass of the amplifier protein used, the mass amplifying strategy leads to significant mass change and sensitive FA detection. Two probes designed based on this strategy against ATP and cocaine were demonstrated to have detection limits of 0.5 μM and 0.8 μM, respectively. More importantly, this strategy is generally

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