Single Molecule Fluorescent Colocalization of Split Aptamers for

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Single Molecule Fluorescent Colocalization of Split Aptamers for Ultrasensitive Detection of Biomolecules Hongding Zhang, Yujie Liu, Kun Zhang, Ji Ji, Jianwei Liu, and Baohong Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01916 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Analytical Chemistry

Single Molecule Fluorescent Colocalization of Split Aptamers for Ultrasensitive Detection of Biomolecules Hongding Zhang, Yujie Liu, Kun Zhang, Ji Ji, Jianwei Liu*, Baohong Liu* Department of Chemistry, Shanghai Stomatological Hospital, State Key Laboratory of Molecular Engineering of Polymers and Institute of Biomedical Sciences, Fudan University, Shanghai 200433, P. R. China * Corresponding author email: [email protected]; [email protected] Phone: 86-21-65641740 ABSTRACT: Single-molecule fluorescence imaging is a promising strategy for biomolecule detection. However, the accuracy of single-molecule method is often compromised by the false-positive events at the ultralow sample levels that are caused by the nonspecific adsorption of the fluorescent labeled probe and other fluorescent impurities on the imaging surface. Here, we demonstrate an ultrasensitive single molecule detection assay based on dual-color fluorescent colocalization of spilt aptamers that was implemented to the measurement of adenosine triphosphate (ATP). The ATP aptamer was split into two fragments and labeled with green and red dye molecules respectively. When the two probes of split aptamers were brought together by the target ATP molecule, the two colors of fluorescence of two probes were simultaneously detected through two channels and projected to the correlated locations in the two halves of image. The colocalizaiton imaging of two split apatamer probes greatly excluded the false detection of biomolecules that was usually caused by the fluorescent noise of single non-bound aptamer probes and impurities, and further improved the accuracy of measurement. The assay showed excellent selectivity and high sensitivity for ATP detection with linear range of 1 pM - 5 nM and a detection limit of 100 fM. This versatile protocol of single molecule colocalization of split apatamer can be widely applied to the ultrasensitive and highly accurate detection of many types of biomolecules in basic research and biomedical applications.

Detection of biomolecules with excellent selectivity and sensitivity makes great contributions to biomedical research, especially molecular diagnostics.1 However, traditional methods for biomolecular detection often suffer from insufficient specificity and sensitivity.2-4 The single-molecule based approach holds the advantages of ultrahigh sensitivity, good selectivity, and low sample consumption.5-8 Moreover, in contrast to the monitoring of ensemble average by conventional methods, single-molecule based methods are able to break the ensemble average and count single molecules with one by one that enables the measurement of sample heterogeneity. 9-12 Given the advantages of single molecule technology, many strategies have been developed to perform the single molecule analytical measurements of biomolecules. For example, the Zhang group established a series of single molecule FRET detection assays of proteins that applied quantum dots (QDs) as the fluorephore to generate bright fluorescent signal.13,14 Similarly, Quake developed a single molecule method to simultaneously detect the multiple epigenetic modifications of genome.15 Beyond the typical single molecule fluorescence imaging, Fang proposed a new strategy to combine the technique of fluorescence recovery after photobleaching (FRAP) in the single molecule fluorescence detection and characterize the dynamics of newly delivered transforming-growth-factor-βtype II receptor on the live cell membrane, that provided more mechanistic insight about the delivery of the transmembrane receptors.16 Such single molecule fluorescence based methods not only possess high sensitivity, but also provide more information about the biomolecules that cannot be observed at a bulk level.15-17

The super high sensitivity of single molecule detection methods can convey big advantage, but it also pays the price of being low tolerant to false-positive events under the condition of ultralow sample concentration that are caused by the unbound fluorophore labeled probes and fluorescent impurities.18, 19 To suppress the false detection of unbound probe and impurity, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) approach20 and competitive DNA-binding scheme21 were adopted to reduce the false positive signal. The former used transiently binding short DNA probes as indicator and analyzed the lifetime of the probebound state that was distinctive under different conditions. Thus it achieved high discrimination against false-positive signal via distinguishing the kinetic signature of the short DNA probes. The latter used trace amount of fluorescent labeled targets to compete for probe ligands with high concentration of unlabeled target to monitor the fraction of hybridized probe DNA, and greatly decreased the background signal. However, the data analysis of these methods was complex and time-consuming. Apart from these methods, the dual-color colocalization was also used for reducing false-positive signal due to its ability to acquire two signals of different colors simultaneously and recognize the nonspecific binding or impurities that only have one color. Balasubramanian and co-workers firstly extended the fluorescent colocalization detection scheme down to single molecule level. The approach dramatically decreased the background signal and thus improved the sensitivity of the DNA detection.22 Recently, Wang’s group reported the use of dual-color colocalization for detection of low abundance protein on the solid phase interface.18 Utilizing dual-color aptamer-functioned QDs as nanoprobes, they re-

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duced the false positive signals significantly and thus improved the detection accuracy. Recently, split aptamers have attracted a lot of attention due to their great superiority in suppressing non-specific adsorption and improving the accuracy of detection.23,24 Although the split aptamers are split from their intact aptamers, their binding affinity with target is still remained. The less secondary structure and shorter sequences of split aptamers make them less prone to nonspecific adsorption.23 Therefore, many biosensing assays based on split aptamers including electrochemistry, fluorescence, and colorimetric methods have been developed for the detection of biomolecules.25-28 Although split aptamers have been used to quantify the degree of aggregation by using single molecule photobleaching technology,29 they have not been used for inhibiting the false positive signal in single molecule fluorescent detection. In this paper, we report a novel strategy of single molecule fluorescent colocalization based on split aptamers for ultrasensitive and highly accurate detection of biomolecules, which significantly avoids the positive false-detection of target molecules. This assay was demonstrated to quantitatively measure ATP with ultralow concentration. The ATP aptamer was split into two DNA fragments according to the previously reported method,25,30 and denoted as probe 1 and probe 2. In the presence of target ATP, a sandwich complex composed of probe 1/ATP/ probe 2 induced by ATP could be formed because of high affinity between ATP and its split aptamers, which brought two fluorescent colors from the two probes together. Then the two fluorescent colors of split aptamers were separately detected through two channels and projected into two bright spots at the correlated locations in the two halves of one image respectively. When the two halves of image were properly aligned, the corrected two spots in two halves corresponding to the probe 1 and probe 2 respectively overlapped to each other. Because the nm-scale distance between two aptamers could not be resolved by optical microscopy. In case the microscope detected false signal from non-bound aptamers or fluorescent impurities that were mixed in the real target molecules, this false signal would be easily recognized because the false signal of mono-color did not have counterpart of the other color in the same location image. In this way, the assay of single molecule dual-color colocalization imaging of split aptamers enables the ultrasensitive detection of ATP with high accuracy.

EXPERIMENTAL SECTION Materials. Adenosine triphosphate (ATP), Cytidine triphosphate (CTP), Uridine triphosphate (UTP), Guanosine triphosphate (GTP), Streptavidin (SA) were purchased from Sangon (Shanghai, China). Poly(L-lysine)-Poly(ethylene glycol)Biotin (PLL-PEG-Biotin) and Poly(L-lysine)-Poly(ethylene glycol) (PLL-PEG) were purchased from Susos AG Inc. (Switzerland). Tris(hydoxymethyl)aminomethane (Tris) was obtained from BoAo Biological Co., Ltd (Shanghai, China). Dithiothreitol (DTT), sodium chloride (NaCl), potassium hydroxide (KOH), phosphate, and ethanol were purchased from Sigma-Aldrich (St Louis, USA). All chemicals were of analytical grade and without further purification. Ultrapure water used in this work was obtained through a Millipore Milli-Q water purification system (Biller, MA, USA). Oligonucleotides labeled with fluorescent dyes were synthesized from Sangon Biological Engineering Technology & Services Co.,

Ltd. (Shanghai, China) and purified by HPLC. The singlestrand DNA strands were confirmed by mass spectrometry and their sequences are listed as follows: Probe 1: 5’-Biotin-TTTTTTTTTTACCTGGGGGA GTAT-Cy3-3’ Probe 2: 5’-TGCGGAGGAAGGT-Cy5-3’ Phosphate buffer saline (PBS, pH 7.4) used in this paper contained 20 mM phosphate and 300 mM NaCl. Tris-HCl buffer (20 mM, pH 8.0) was prepared with 20 mM MgCl2, 10 mM NaCl and 2 mM DTT. The mixture of PLL-PEG and PLLPEG-Biotin and the probe 1 were prepared in 20 mM of PBS. The probe 2 and ATP were prepared in Tris-HCl buffer. Coverslip Pretreatment. Coverslips (Fisher, 25 mm × 25 mm) were cleaned as following: The coverslips were firstly washed in 1 M of KOH solution by an ultrasonic bath for 10 min to remove grease for three times. After being ultrasonicated in ultrapure water for 10 min, the slides were ultrasonically cleaned in ethanol for 10 min. Then, the coverslips were washed by ultrapure water again for another 10 min with three times. Finally, the slides were dried by N2 stream. Preparation of sample cells and the surface modification of the coverslip. The sample cell was composed of a coverslip and a glass slide (25 mm × 40 mm, 6 mm) with a hole in the middle, the diameter of the hole was 5 mm. The cell was constructed by gluing the coverslip on the bottom of the hole of the glass slide via vacuum grease. Then, the coverslips were passivated with PEG according to the previously reported methods with a little modification.31,32 Briefly, the sample cells were etched in vacuum plasma (PDC-002, Harrick Plasma Inc., U.S.) for 10 min followed by incubating with a mixture of PLL-PEG and PLL-PEG-Biotin (10: 1) for 1 h. After that, the cell was washed by PBS buffer for three times to remove excessive PLL-PEG-Biotin. Then, 50 µL 0.2 mg/mL of SA was added into the sample cell and incubated for 15 min to bind the biotin. After that, the sample cell was washed with PBS buffer for three times. The modified sample cell added with 50 µL of PBS buffer was stored at 4 ˚C for further use. The Detection of ATP. The ATP detection process was described as follows: Briefly, the SA modified sample cell was first incubated with 50 µL 0.5 pM of probe 1 solutions for 15 min, then washed by Tris-HCl buffer for three times to get rid of unbounded probe 1. Subsequently, 25 µL 20 nM of probe 2 solutions and 25 µL of ATP solutions were added into the sample cell, and the mixed solution was incubated at 37 ˚C for 45 min. Afterward, the cell was washed with Tris-HCl buffer for three times to wash away the unbounded probe 2 and ATP molecules on the surface of the coverslip. Finally, the sample was introduced with 50 µL of Tris-HCl buffer for single fluorescence imaging. Selectivity of the proposed assay. 1000 nM of GTP, UTP, CTP were respectively added into the reaction system instead of 10 nM of ATP and incubated at 37 ˚C for 45 min. After that, all the samples were washed with Tris-HCl buffer for three times. Finally, the samples were added into 50 µL of Tris-HCl buffer for single fluorescence imaging. Detection of Real Samples. The human serum samples obtained from Fudan University Shanghai Cancer Center was used for the complex sample detection. The samples were diluted 10000-fold with Tris-HCl buffer for ATP detection. The practical samples were spiked with various concentrations


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Analytical Chemistry of ATP. The amounts of ATP in the human serum samples and diluted samples were analyzed by the proposed assay. Single Molecule Colocalization Imaging. All the single molecule experiments were carried out in a home-built single molecule fluorescent imaging system that was based on a wide field epi-fluorescence setup. The 532 and 632 nm Laser beams were focused by a wide-field lens and then collimated by an oil immersion objective (NA 1.45, 100 ×, Olympus, Japan) to form the circular excitation fields with radiuses of tens micrometers on the coverslip surface that exited the Cy3 and Cy5 fluorophores respectively. The fluorescence from Cy3 and Cy5 was collected by the same objective and then separated and steered onto the two halves (green channel and red channel) of an EMCCD camera (Ixon DU897, Andor Technology Plc., U.K.) by using beam splitter (Optosplit II, Andor Tech-

nology Plc., U.K) respectively. For each sample, we imaged 10 regions and each region was imaged 60 successive frames with an exposure time of 100 ms. All the measurements were performed at room temperature. Analysis of Single Molecule Fluorescence Images. The MATLAB (Mathworks Inc., MA, U.S) program was employed to analyze the single molecule image. For each frame, the size of 40 x 20 µm was used as the analysis area. Firstly, the coordinates of bright spots of both channels were determined, respectively. Secondly, the Cy3 spots were counted according to the coordinates found in the first step, and denoted as Ncy3. At last, all the spots in two channels were compared and matched with certain criteria where the number of matched Cy3 and Cy5 pairs was referred as Nco.

Scheme 1. Scheme illustration of single molecule dual-color colocalization assay for ATP detection based on split aptamers

RESULTS AND DISCUSSION Detection of Target ATP based on Single Molecule Fluorescent Colocalization of Split Aptamers. The principle of the assay is shown in scheme 1 using ATP and its split aptamers as a model. The surface of coverslip is firstly functionalized with positive PLL-PEG-Biotin via electrostatic adsorption for the passivation and biotinylation of slip surface. Subsequently, SA is immobilized on the slip surface through the specific binding between SA and biotin. The probe 1 is labeled with Cy3 at 3’ terminal and biotin at 5’end, and can be anchored at the coverslip surface through the specific binding between SA and biotin. The probe 2 is designed with Cy5 being labeled at 3’ terminal. Because of the stochastic combination of the probe 1 on the coverslip, the counts of Cy3 from probe 1 in various regions are slightly different, which may lead to the discrepancy of the pairs of the dual-color colocalization. Therefore, to ensure the accuracy of the ATP quantification, the ratio value (δ) is applied for the quantification of ATP concentration. The δ value was calculated as following:

δ=

N Co N Cy3

(1)

Nco represents the colocalization pairs of double colors; NCy3 stands for number of Cy3 counts from probe 1. In the presence of ATP, a sandwich complex with probe 1/ATP/ probe 2 induced by ATP can be formed, which brings the two colors together. Herein, the dual-color of the sandwich complex can be imaged by colocalization, leading to the high value of δ. In the absence of target, only one color (green or red) can be imaged, which results in the small value of δ. Therefore, the target ATP can be detected by this principle. The experimental setup of dual color fluorescence imaging refers to the experimental section. Based on single molecule imaging, this assay holds high sensitivity for the detection of target molecule. The feasibility of the single molecule based ATP detection was demonstrated as shown in the Figure 1. Many spots from the Cy3 can be observed at green channel in Figure 1A, this is because many probe 1 molecules are assembled on the coverslip surface via specific combination between SA and biotin. In the absence of target ATP, large amount of Cy3 fluorescence imaging counts can be observed, only few Cy5 fluorescence imaging counts caused by nonspecific adsorption are observed in the red channel (Figure 1B). The results indicate that the false positive signal caused by nonspecific adsorption is very little. While in the presence of target ATP, both Cy3 and Cy5 fluorescent imaging signals are observed simultane-



ously, this is because many complexes of probe 1/ATP/ probe 2 induced by ATP are formed on the surface of coverslip (Figure 1C). The average δ value of that in the presence of ATP is much higher than that in the absence of ATP (Figure S1). These results demonstrate that this proposed approach for ATP detection is feasible. Additionally, almost all the spots showed clear photobleaching and photoblinking with a single step in their intensity trace (Figure S2), demonstrating that each fluorescence spot corresponded a single Cy 3 or Cy5 dye molecule.

Figure 1. Single molecule fluorescence imaging from Cy3 and Cy5 on the surface of coverslip, respectively. (A) PLL-PEGBiotin + SA + Probe 1; (B) PLL-PEG-Biotin + SA + Probe 1 + Probe 2; (C) PLL-PEG-Biotin + SA + Probe 1 + ATP + Probe 2. Probe 1: 0.5 pM, Probe 2: 10 nM. ATP: 100 nM. Scale bar: 5 µm.

Figure 2. Determination of fluorescent impurity and non-specific binding of probes in the absence of probes or effective tethering of probes to coverslips. Panel a and d: PLL-PEG-Biotin; b and e: PLL-PEG-Biotin + Probe 1; c and f: PLL-PEG-Biotin + Probe 2; Probe 1: 0.5 pM, Probe 2: 10 nM. Scale bar: 5 µm. Single-molecule based methods usually suffer from the false-positive events at the ultralow sample levels and thus the accuracy of detection is compromised. Because the false positive signal was mainly caused by non-specific adsorption

when the slide surface was very clean, the non-specific adsorption of the assay was thus investigated. As shown in Figure 2, no count is observed at both green channel and red channel on the PLL-PEG-Biotin modified surface of slide, indicating that the surface is very clean and can not affect the single molecule imaging (Figure 2a and Figure 2d). Only few counts are observed in the Figure 2b and Figure 2f, suggesting that nonspecific adsorption of probe 1 and probe 2 on the coverslip surface is little. The low background may be ascribed to the excellent passivation of PEG31 and the short sequence of the split aptamers that are not easy to attach to the slide surface at the low concentration of probe 2 molecules33. Optimizing the Distribution Intensity of Split aptamers. The distribution of the probe 1 assembled on the coverslip surface greatly determines the sensitivity of ATP detection. Although the high distribution intensity of capturing probes on a coverslip benefits the capturing of target molecules and improves the sensitivity of assay, the too dense distribution of it will hamper the distinction of fluorescent spots from each other in the image and causes error of molecule counting. The optimization results of concentration of capturing probe show that 1 or 5 pM generates a too dense distribution of capturing probe, but 0.05 and 0.1 pM do not generate sufficient amount of capturing probe on a coverslip (as shown in Figure 3A). Thus, 0.5 pM is relatively an optimizing concentration generating a suitable intensity and uniform distribution of the probe 1. The concentration of probe 2 also plays an important role in the detection of ATP. Because of the very low intensity of the probe 1 on a coverslip and very low concentration of target molecules, probe 2 needs to be added to the solutions as much as possible in order to form enough sandwich complex of probe 1/ATP/probe 2. Though adequate probe 2 can increase the probe target interaction rates,34 too much excess of probe 2 would cause badly nonspecific adsorption that raises the probability to mis-match a pair of unbound but very close probe 1 and probe 2 and treats them as one bound complex leading to a false-positive count (as shown in Figure 3B). With optimizing, 10 nM of probe 2 is determined to be the suitable concentration for subsequent experiments. Characterization of the Single Molecular Detection Performance. A series of ATP samples with various concentrations are measured to evaluate the sensitivity of the proposed assay. To avoid the sampling error,35 the average δ value for 10 regions of each sample is used for quantifying the ATP concentration. Figure 4A plots the measured δ values vs. the concentrations of ATP samples. In the low level of the ATP content, the δ value increases greatly with the concentration of the ATP, while in the high level of ATP concentration, the δ value increases much slowly. This is because the amount of ATP combined on the surface of coverslide tends to equilibrium at high concentrations.36-38 In the range of 1 pM - 5 nM ATP, the δ values show a good linear correlation with the logarithm of ATP concentrations (as shown in Figure 4C) and the plot can be fitted by equation δ = 0.1782 + 0.0384 lgC (R2=0.9726), and the detection limit is 100 fM (Figure 4A and inset of Figure 4B). It is worth noting that the sensitivity of this proposed assay has improved 106 and 103 fold respectively, compared with the reported electrochemical sandwich assays25 and nanoprobe-enhanced split aptamer-based electrochemical sandwich assays(NE-SAESA)39. Such high sensitivi-


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Analytical Chemistry ty comes from the capability of this assay to detect and count

single

molecules.40,41

Figure 3. (A) Influence of the probe 1 concentration. The concentrations of probe 1 are 5 pM, 1 pM, 0.5 pM, 0.1 pM, 0.05 pM (from left to right), respectively; (B) Influence of the probe 2 concentration. The concentrations of probe 2 are 100 nM, 50 nM, 10 nM, 5 nM, 1 nM (from left to right), respectively. Scale bar: 5 µm.

Figure 4. (A) Single molecule fluorescence imaging from Cy3 and Cy5. (B) δ values calculated with different concentrations of ATP from 100 fM to 10 nM (inset data are δ values at low concentrations of ATP); (C) Liner relationship between δ values and logarithm of ATP concentration. (D) The binding curve of split aptamer with ATP. The error bars represent the standard deviation of three repetitive experiments. Scale bars: 5µm. The binding interaction of ATP with its split aptamers is further investigated. When the concentration of probe 2 is greatly larger than probe 1, the influence of the interaction between ATP and its split aptamers can be considered to only depend on the concentrations of ATP and probe 1. Therefore, the binding constant (K) of the ATP and its split aptamers is calculated according to the following equation.42,43

r=

K i [C free ] [C bound ] m = ∑ ni [ p total ] 1 + K i [C free ] i =1

(2)

trations of total split aptamers, bound ATP and free ATP that is not bound on the slip surface, respectively. Because the amount of ATP molecules bound to the slide surface is very little under the condition of single molecule level, [Cfree] can be approximately considered as the concentration of the added ATP ([ATP]). r stands for the fraction of bound ATP molecules per aptamer. Since the δ value is equivalent to the ratio of the bound ATP and probe 1, r can be replaced by δ. Assuming that the interaction between ATP and its aptamers is a single class of binding sites, the above equation can be simplified as following:

Here, Ki and ni refer to the binding constant and the number of sites of class i. [Ptotal], [Cbound] and [Cfree] stand for the concen-



r=n

K[ ATP] 1 + K[ ATP]

(3)

Figure 5. (A) Overlap of the single molecule fluorescent images of probe 1 and probe 2 in the prescence of CTP, GTP, UTP, ATP, respectively. (B) δ value in the presence of 10 nM ATP and 1000 nM other analytes. The error bars represent the standard deviation of three repetitive experiments. Scale bar: 5 µm.

Therefore, the binding constant K and number of binding sites n can be calculated through the fit of binding curves (Figure 4D) with Eq. (3), and were worked out to be 602.8650 nM-1 and 0.1340 respectively. Furthermore, the selectivity and the binding specificity of the proposed method for ATP detection are assessed. The system is incubated with ATP and its analogues such as GTP, UTP and CTP. And then the single molecule imaging of the system incubated with ATP and its analogues are performed respectively (Figure 5A). As shown in Figure 5B, the changes of δ values resulted from GTP, UTP, CTP are less than 15% of that of ATP, even if their concentrations are 100-fold higher than that of ATP. This is possibly due to the high affinity between ATP and its aptamers, and less non-specific adsorption. These results demonstrate that such split aptamer-based approach has good selectivity for ATP analysis. Detection of ATP in Human Serum. The proposed single molecule imaging based assay is used for the detection of ATP in human serum samples. The ATP level in human serum is about 1 µM. Therefore, 10000-fold dilution of human serum is necessary. By taking advantage of standard addition method, the concentration levels of ATP in two human serum samples are detected to be 82 pM and 79 pM respectively, that are approximate to the values of previously reported methods.44,45 After the diluted human serum samples are spiked with different ATP samples, the detection recoveries of every samples are shown in Table S1, indicating that the accuracy of the proposed method is acceptable. These results demonstrate that the method of single molecule dual-color colocalization can be applied for the detection of target molecules in practical samples.

sensitivity than the typical methods. The ultrahigh sensitivity of the single molecule fluorescence method is usually accompanied with poor accuracy because the false-positive events become severe under the detecting level of single molecules. The proposed method recognizes the single target molecule by judging the spatial overlapping, i.e. co-localization, of two orthogonal fluorescence-color labelled split aptamers to avoid the mis-determination of unbound probes and impurities effectively. So the good accuracy as well as ultra-high sensitivity can be achieved. The concept-approve application of such protocol on the human serum samples implies its practicability. Moreover, provided by the flexibility of split aptamer to bind varying target molecules, this strategy can serve as a general platform to detect many kinds of biomolecules in basic research and clinical testing.

CONCLUSIONS

ACKNOWLEDGMENT

In summary, an ultrasensitive and specific assay based on the single molecule dual-color fluorescent colocalization of split aptamer has been developed. The application of single molecule fluorescence imaging to count single target molecule in this approach achieves a lower detection limit of 100 fM in the case of ATP that provides 3 ~ 6 orders of magnitude higher

This work was supported by NSFC 21775028, 21375028, STCSM (16391903900, 17JC1401900), and Collaborative Innovation Center of Chemistry for Energy Materials.

ASSOCIATED CONTENT Supporting Information The average δ value in the absence of ATP and presence of ATP, represent intensity traces of single green dye and red dye molecule, and Recoveries of human serum samples detection in this study. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected]. Fax: +86-21-65641740

Notes The authors declare no competing financial interest.

REFERENCES (1) Ma, F.; Li, Y.; Tang, B.; Zhang, C. Acc. Chem. Res. 2016, 49, 1722-1730.


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