Water as a Universal Infrared Probe for Bioanalysis in Aqueous

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Water as a Universal IR Probe for Bioanalysis in Aqueous Solution by Attenuated Total Reflection Surface Enhanced Infrared Absorption Spectroscopy Yang Liu, Wen-Jing Bao, Qian-Wen Zhang, Jin Li, Jian Li, Jing-Juan Xu, Xing-Hua Xia, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03659 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Analytical Chemistry

Water as a Universal IR Probe for Bioanalysis in Aqueous Solution by Attenuated Total Reflection - Surface Enhanced Infrared Absorption Spectroscopy

Yang Liu, Wen-Jing Bao, Qian-Wen Zhang, Jin Li, Jian Li, Jing-Juan Xu, Xing-Hua Xia,* and Hong-Yuan Chen

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China *To whom correspondence should be addressed. E-mail: [email protected]

ABSTRACT: Monitoring the properties and reactions of biomolecules at interface have attracted ever-growing interest. Here, we propose an approach of infrared analysis technique that utilizes water molecule as a universal probe for in situ and label free monitoring interfacial bioevents in aqueous solution with high sensitivity. The strong infrared (IR) signal of O-H stretching vibrations from the repelled water is used to sensitively reveal the kinetics of interfacial bioevents at molecular level based on the steric displacement of water using an attenuated total reflection - surface enhanced infrared absorption spectroscopy (ATR-SEIRAS). Using interfacial immunorecognition and DNA hybridization as demonstrations, water IR probe offers 26 and 34 times higher sensitivity and even 200 and 86 times lower detection limit for immunosensing and DNA sensing, respectively, as compared to the traditional IR molecular fingerprints. Keywords: water IR probe, ATR-SEIRAS, steric effect, immuno-recognition reaction, DNA hybridization

Attenuated total reflection - infrared absorption spectroscopy (ATR-IRAS) has been considered as a powerful tool for molecule-level study of interfacial structures and 1

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processes in aqueous solutions owing to the confined penetration electric field which significantly suppresses the interference from bulk water.1-4 Recent studies have revealed that the analytical sensitivity of this technique can be significantly improved by introducing surface enhanced substrates with localized surface plasmon resonance wavenumber located in the mid-IR region, named as attenuated total reflection - surface enhanced infrared absorption spectroscopy (ATR-SEIRAS). This ATR-SEIRAS has been successfully used to study the interfacial behaviors,5-9 the interfacial biomolecular recognition events,10-17 the relationship between conformation and function of redox proteins under potential control,18-21 and the influence of membrane potential on membrane proteins in situ and real time.22,23 Although great progresses have been achieved in this research area,24,25 this new technique yet suffers from the interference from the abundant existence of solvent water at the surface since water molecules are of the tremendous IR absorption cross-section.26 Thus, quantitative analysis in subnanomolar level or even lower levels of the targets at the IR enhanced interfaces with limited enhancement factor cannot be easily achieved only based on the IR features of the targets in aqueous solutions. Interfacial processes including adsorption/desorption, chemical/biochemical reactions occurring at surfaces are accompanied by change in number of water molecules due to the steric effect since any species with defined structure or conformation has corresponding volume. Thus, we propose that water, with the great IR absorption cross-section, could be considered as a universal IR probe to sensitively monitor interfacial processes occurring at the enhanced substrate. There are a few works qualitatively studying the interactions between nanoparticles and proteins and lipid membrane using IR signal of water molecules.23 A quantitative method suitable for monitoring different sized interfacial molecules along with interfacial events using water as the IR probe has not yet been proposed, measured and evaluated. Herein, we propose an ATR-SEIRAS approach to the qualitative and quantitative monitoring of interfacial processes using water molecule as a universal IR probe. First, this work illuminates that this method is closely related to the steric effect of the interfacial molecules to be detected. In this work, comparing the IR signal of the 2


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aqueous solution before and after polystyrene (PS) spheres assembled at the enhanced substrate, respectively, we find that repelled water shows sensitive IR signal related to the interfacial variation. Then, we utilize DNA hybridization and macromolecular immuno-recognition as demonstrations to verify the universality of using water molecule as IR probe in quantitative monitoring of interfacial processes in situ and real time. Both of the processes can be detected effectively by ATR-SEIRAS because the sizes of reacting biomolecules are within the effective penetration depth at the Au nanoparticles substrate.7,27-30 Results show that water molecule at the enhanced substrate is a promising and universal IR probe for sensitively monitoring interfacial events and it will expand the scope of IR applications in chemical, biological and medical sciences.

EXPERIMENTAL SECTION Materials and Reagents. ZnSe semispherical prism with 20 mm in diameter was purchased from Bosheng Quantum Technology (Changchun, China). Emulsions of 1.0% in mass fraction of polystyrene (PS) spheres with 465 nm in diameter were bought from Suzhou Micro Technology Co., Ltd. (China). Bovine serum albumin (BSA), mercapto hexanol (MCH), mercaptopropionic acid (MHA), 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (USA). Rabbit IgG and Goat anti-Rabbit IgG were bought from Wuhan Boster Biological Engineering Co., Ltd. (China). Target DNA (5'GGT AAG CAA CTG ATT-3') and probe DNA (5'-SH-(CH2)6-AAT CAG TTG CTT ACC-3') were purchased from Shanghai Sangon Biotech Co., Ltd. (China). All reagents were of analytical grade and used as received. All aqueous solutions were prepared from ultra-pure water (> 18 MΩ • cm, Millipore, USA). Instrumentation. Infrared spectra of averaged 128 scans in a wavenumber range from 4000 cm-1 to 650 cm-1 with a resolution of 4 cm-1 were collected at 25 oC with a Nicolet 6700 Fourier transform spectrometer (Thermo Fisher, USA) equipped with a liquidnitrogen-cooled MCT detector. The morphology of the gold enhanced substrate prepared on the ZnSe slice was characterized by atomic force microscope (AFM, 3



Agilent-5500, USA). The morphology of the assembled PS spheres on gold nanoparticles film/ZnSe slice was characterized by a scanning electron microscopy (SEM, Hitachi-S4800, Japan) at an accelerating voltage of 5 kV. Preparation of surface enhanced substrate. The preparation of gold nanoparticles enhanced substrate on a flat ZnSe prism was recently reported in our group.1 Before deposition, surface of the semispherical ZnSe ATR prism was polished with aluminum oxide powder (1 μm in diameter) for several minutes and rinsed subsequently by sonication in anhydrous ethanol and ultrapure water. After this cleaning process, a drop of 10 mM HAuCl4 aqueous solution was casted on the ZnSe prism surface at 40 oC for 25 s. The reaction was terminated quickly by washing with large amount of ultra-pure water. A layer of gold nanoparticles film was successfully deposited on the ZnSe surface. The structure of the gold nanoparticles substrate was imaged by AFM. AFM image of the gold nanoparticles substrate. Sample for AFM image was prepared as follows: a layer of gold nanoparticles film was deposited on a slice of ZnSe optical window using the same method as for ZnSe prim. Fabrication of model system. On the gold nanoparticles film/ZnSe, a single layer of close-packed PS spheres was self-assembled from a PS colloids suspension for overnight (ca. 12 h). After drying, an infrared background spectrum was collected. Sample IR spectra were collected after 400 μL water added into the sample container. In control group, background and sample spectra of the gold nanoparticles film/ZnSe without assembly of PS spheres were collected in the absence and presence of 400 μL water, respectively. The structure of the assembled PS spheres on gold nanoparticles film/ZnSe slice prepared by the same method as for ZnSe prism was imaged by SEM. Monitoring of immuno-recognition reaction. The gold nanoparticles film/ZnSe was first modified with a self-assembled layer of mercapto acid from a solution of 400 μL 10 mM mercapto acid aqueous solution for overnight (ca. 12 h). After rinsing the surface with ultrapure water, 400 μL 25 mM EDC/NHS (1:1) solution was added onto the surface for 30 min to activate the carboxylic groups. This activation process was terminated by washing with abundant water. Then, 200 μL 20 μg/mL Rabbit IgG solution was added to the activated surface and incubated for 1 h for covalent bonding 4


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of the protein to the gold nanoparticles film via the formation of amide bonds. This surface was further passivated by using 200 μL 1% BSA for 10 min to remove the physically bounded proteins. After the surface was washed with ultrapure water, 200 μL PBS (50 mM, pH=7.4) was added and a background spectrum was recorded. The sample spectra for immunoreaction were collected after adding 200 μL 20 μg/mL Goat anti-Rabbit IgG into the reaction container. All the spectra were obtained at 25 oC. Measurement of DNA hybridization. A self-assembled layer of probe DNA was first covalently assembled on a gold nanoparticles film/ZnSe surface via the Au-S bonds from an aqueous solution of 100 μL 1 μM probe DNA containing 1 M NaCl

for

overnight. This surface was then passivated by MCH from an aqueous solution of 100 μL 0.3 mM MCH for 30 min. After the surface was washed with 1 M NaCl aqueous solution, a background spectrum of 100 μL 1 M NaCl aqueous solution was collected. Sample spectra for target DNA was recorded after addition of 100 μL 1 μM target DNA aqueous solution containing 1 M NaCl into the reaction container. All the spectra were obtained at 25 oC.

RESULTS AND DISCUSSION To demonstrate the feasibility of water molecule as the quantitative signal probe in Infrared spectroscopic analysis, a deposited monolayer of PS spheres on the gold nanoparticles film/ZnSe was used as a model since the steric effect from the displacement of water by assembling PS spheres would be notable as illustrated in Figure 1a. The spectrum (Figure 1b, green curve) of water molecules on the gold particles film without PS spheres shows a significant band centered at ca. 3400 cm-1 of O-H stretching vibrations (including symmetric and antisymmetric vibrations) and a small band at ca.1670 cm-1 of O-H bending vibration as usually observed. Thus, we use the IR signal of O-H stretching vibrations to describe the displacement effect of water at the interface. A compacted monolayer of PS spheres is first deposited on the gold nanoparticles film/ZnSe as evidenced by the SEM image (Figure 1b, inset). Since the volume of a PS sphere (diameter≈465 nm) is about 109 5



times larger than that of H2O (diameter≈0.3 nm), the spectrum of PS/Au NPs/ZnSe in the presence of water shows a significant decrease in the peak intensity of O-H stretching vibrations (Figure 1b, pink curve). The differential spectrum (Figure 1b, red curve) of the two states shows the negative bands of the repelled water molecules (OH stretching vibrations centered at ca. 3400 cm-1; and O-H bending vibration at ca. 1640 cm-1), and the positive bands of the deposited PS spheres (C-H symmetric and antisymmetric stretching vibrations at 2844 cm-1, 2933 cm-1 and 3020 cm-1, respectively). It is clear that the IR signals of the displaced water are much stronger than those for the deposited PS spheres since the band integral of O-H stretching vibrations from the displaced water molecules is more than 100 times of the band integral of C-H stretching vibrations from the deposited PS spheres. Thus, the strong IR signal of the repelled water can be a sensitive probe to characterize the interfacial events, and the integral of O-H stretching band from the repelled water provides the quantitative IR characterization. Based on the results, we can conclude that water molecules can be used as the sensitive IR probe for quantitatively and qualitatively monitoring interfacial events. To demonstrate the universality of water molecule as the sensitive IR probe, two interfacial bio-events with different molecular sizes, e.g., immune-recognition reaction and DNA hybridization were performed.

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Figure 1. (a) Scheme illustrating the interface for obtaining displaced water by deposited PS spheres on gold nanoparticles film/ZnSe by using the surfaces of Au NPs/ZnSe (state ①) as reference and PS/Au NPs/ZnSe (state ②) as sample. (b) Spectra obtained from the surface of states ① and ②. The differential spectrum of these two states is also displayed as the red curve. Inset: The SEM image of the deposited PS spheres on Au NPs film/ZnSe slice.

Monitoring of immuno-recognition reaction. Since a great majority of the interfacial bioevents of proteins can be detected effectively at Au enhanced substrate by ATRSEIRAS,7,27-30 the interfacial immuno-recognition reaction is first demonstrated to inspect the universality of water molecule as the sensitive IR probe for biomacromolecule. The antigen Rabbit IgG is previously assembled on gold nanoparticles film via the Au-S covalent bond. After passivation with bovine serum albumin (BSA) to remove the nonspecific adsorbed proteins, a spectrum of this antigen assembled interface is collected as the reference. When the antibody Goat anti-Rabbit IgG is added, immnuo-recognition reaction occurs (illustrated in Figure 2a). This recognition reaction is monitored by water molecule probe. Figures 2b and c show the evolution of IR spectra of the recognition reaction with time.

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Figure 2. (a) Scheme illustrating the immuno-recognition reaction. Evolution of the infrared segmented spectra of (b) the O-H stretching bands from the repelled water and (c) the amide bands from the bounded protein vs. reaction time in a solution of 10 mg/L antibody + 50 mM PBS (pH 7.4). (d) Integrals of the bands for O-H stretching vibrations from the repelled water and amide I band from the bounded protein vs. reaction time. (e) Plot of ln(1−θ) vs. time and the corresponding fitting lines, where θ is the fractional coverage of surface immobilized antigen. (f) Correlation between the integrals of water and protein bands.

The significant negative bands of O-H stretching vibrations increase with the reaction time (Figure 2b) owing to the continuous displacement of water molecules from the interface by the affinity reaction of antibody to the assembled antigen, while the smaller 8


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amide I band from proteins accordingly increases with time (Figure 2c). Both the signals for the displaced water molecules and the bounded proteins show fast changes at the initial reaction stage and then reach steady-state at ca. 35 min, demonstrating the achievement of reaction equilibrium. For clarity and quantitative analysis, the integrals of the water band and the amide I band are plotted in Figure 2d. It is clear that the increase in water signal is much larger than that for protein signal, showing the importance in interfacial analysis using the displaced water as IR probe. The kinetics of the immuno-recognition reaction is evaluated by a traditional twocompartment model31 assuming that the intramolecular interaction among the goat antibody molecules bound on the antigen can be neglected (detailed description of the calculations refer to the supporting information).15 As shown in Figure 2e, plots of ln(1θ) obtained from the displaced water and the bounded protein signals as function of reaction time present two straight lines, respectively. From the slopes, the antigenantibody binding rate constants are both calculated as 1.5 x 104 M-1s-1 based on the displaced water and bounded antibody signals, respectively. The calculated binding rate constants for antigen Rabbit IgG and Goat anti-Rabbit IgG from displaced water and protein signals are in good agreement with each other and with the ones reported previously,15,16 demonstrating the reliability of the present strategy using the displaced water as IR probe in studying of the interfacial immuno-recognition reaction. The reliability is also confirmed by the linear correlation between the signals of displaced water and bounded protein (Figure 2f). The slope of Figure 2f demonstrates that the detection sensitivity using the displaced water probe is ca. 26 times higher than using the bounded protein signal.

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Figure 3. ATR-SEIRAS segmented spectra of (a) the O-H bands from the replaced water and (b) the amide bands from the bounded protein collected at 35 min of immunoreaction time after adding different antibody concentrations of 0 nM, 2.2 nM, 6.1 nM, 11.6 nM, 19 nM, 34 nM, and 46 nM. (c) Dependence of band integral obtained from the displaced water and the bounded protein, respectively, on antibody concentration of 0 nM, 0.01 nM, 0.03 nM, 0.05 nM, 0.15 nM, 0.36 nM, 0.63 nM, 0.76nM, 2.2 nM, 6.1 nM, 11.6 nM, 19 nM, 34 nM, and 46 nM. The fitting curves follow the Langmuir adsorption isotherm. Inset: amplification of the low concentration range of the dependence of H2O signal on target DNA concentration: 0 nM, 0.01 nM, 0.03 nM, 0.05 nM, 0.15 nM, 0.36 nM, 0.63 nM, and 0.76 nM. (d) Spectral comparison of immune response (10 mg/L antibody + 50 mM PBS, pH 7.4) and non-immune response (10 mg/L BSA + 50 mM PBS, pH 7.4) collected at 35 min of immuno-reaction time.

The evolution of IR spectra as a function of antibody concentration was also studied and the segmented results are displayed in Figure 3a and b. The integrals of the displaced water and bounded antibody bands from the IR spectra at equilibrium reaction 10


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time of 35 min as functions of antibody concentration are shown in Figure 3c. They increase rapidly at low concentrations, increase slowly at middle concentrations, and finally reach steady-state. The inset of Figure 3c gives a linear relation between the probe signal and the low concentration of target antibody. These data can be well fitted using the Langmuir adsorption isotherm as reported in our previous work.15,16 From the fitting, the dissociation constant (KD) of the immune-recognition reaction is obtained as 1.5x10-8 and 6.4x10-8 M from the data of the displaced water and bounded antibody, respectively (detailed information refers to the supporting information, Table S1). The limit of detection calculated from the displaced water signal is 0.01 nM, which is about 200 times lower than that from the bounded protein signal based on 3 times of signal to noise. All the kinetic and thermodynamic parameters obtained from the displaced water signals are well correlating to the ones from the bounded protein signals, demonstrating that the reliability and high sensitivity of the present strategy using displaced water as IR probe when compared to the ones using the traditional IR signals of the targets. Besides, Figure 3d provides the evidence of another advantage that water probe has good selectivity of immune response when compared to non-immune response at equilibrium reaction time of 35 min.

Monitoring of DNA hybridization. To further evaluate the universality of water molecule as IR probe in monitoring of interfacial events, hybridization of DNA with smaller molecule size was studied (schemed as Figure 4a). The capture DNA is first self-assembled on the gold nanoparticles film via Au-S bond. After collection of a background spectrum as the reference, target DNA is added into the system for hybridization, and then sample spectra are recorded. Figure 4b is the ATR-SEIRAS spectra obtained at 130 min of hybridization time. It shows the significant negative band of O-H stretching vibrations at ca. 3400 cm-1 from the displaced water, the small bands of C=O bond of DNA bases between 1662-1732 cm-1,32 which are overlapped with the band of O-H bending vibration at ca. 1637 cm-1, and the middle strong positive bands at 1217, 1080 and 967 cm-1 for phosphate backbone from DNA structure (Figure 11



4b and Table S2, supporting information).33-35 It is clear that the IR band of PO2

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-

stretching vibrations is more significant than the signals of DNA bases. Thus, we use -

the band of PO2 symmetric stretching vibration (ca. 1080 cm-1) to reflect the DNA hybridization process rather than using the DNA base bands since there is a defined correlation between the numbers of phosphate groups and DNA bases.

Figure 4. (a) Scheme illustrating the DNA hybridization. (b) ATR-SEIRAS spectra of DNA hybridization in a solution of 0.5 µM target DNA + 1 M NaCl at 130 min of hybridization time. The negative O-H stretching band at ca. 3400 cm-1 in the sample spectrum represents the displaced water signal, while the positive IR bands of phosphate backbone from DNA structure (ca. 1080 cm-1) represent DNA signal. (c) -

Evolution of the integrals of the PO2 symmetric stretching band and the O-H stretching bands multiplied by -0.05 fold vs. hybridization time. (d) Plot of ln (1−θ) versus time and the corresponding fitting lines, where θ is the fractional coverage of surface 12


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immobilized capture DNA. The equilibrium time is about 130 min. (e) Correlation between the integrals of water and phosphate bands. The slope of the fitting line indicates that the approach using water signal is ca. 34 times more sensitive than using phosphate signal. (f) Dependence of the integrals of the band owing to O-H streching -

vibrations from the repelled water and the band from PO2 symmetric stretching vibration collected at 130 min of hybridization time on target DNA concentration: 0.2 nM, 0.7 nM, 1.2 nM, 2.3 nM, 3.7 nM, 6.4 nM, 15.5 nM, 33.0 nM, 66.3 nM, 145.2 nM and 294.5 nM, respectively. The LOD of water signal is 0.2 nM while the LOD of DNA signal is 15.5 nM. Inset: amplification of the low concentration range of the dependence of H2O signal on target DNA concentration of 0.2 nM, 0.7 nM, 1.2 nM, 2.3 nM, 3.7 nM, respectively. (g) Spectral comparison of the reaction between complementary DNA and mismatched DNA collected at reaction time of 130 min.

Figure 4c shows the evolution of the integrals from the O-H stretching bands -

(black curve) and PO2 symmetric stretching bands (red curve) vs. hybridization time, reflecting the interfacial DNA hybridization kinetics from a solution of 0.5 µM target DNA + 1.0 M NaCl. The ln(1-θ) obtained from the band integrals of displaced water and phosphate groups shows quasi-linear relationship with the hybridization time, respectively (Figure 4d), since the kinetics of DNA hybridization can be described by the traditional two-compartment model.17 The DNA-DNA binding rate constant of 5.2 x 102 M-1s-1 calculated based on water signal is comparable to the one (4.7x102 M-1s-1) from phosphate signal. These values are at the same level of magnitude, implying the reliability of the water probe approach. According to these kinetic curves, after about 130 min the hybridization is getting balanced approximately. Although the real reason for such significant fluctuations (parallel data of almost the same fluctuations displayed in Figure S2, supporting information) of water signal

during DNA hybridization is

not yet clear, the fluctuations between the signals of the displaced water molecules and the bounded phosphate groups are positively correlated as indicated by a quasi-linear relationship (Figure 4e), demonstrating the characteristics of interfacial DNA in 13



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hybridization and the reliability of using water probe in monitoring small sized biomolecules. The slope of Figure 4e indicates that the detection sensitivity using the displaced water probe is ca. 34 times higher than using DNA signal. At equilibrium hybridization time of 130 min, the evolution of the integrals of the -

O-H stretching band from the displaced water and the PO2 symmetric stretching band from the bounded DNA as function of target DNA concentration was monitored as well and the results are shown in Figure 4f. As expected, the signals of the displaced water and the DNA rapidly increase at low concentrations, increase slowly at middle concentrations, and finally reach steady equilibrium. The inset of Figure 4f gives a linear relation between the probe signal and the low concentration of target DNA, which is close to that of other reported DNA sensor.36 Herein, the water probe shows a linear range under 4 nM with a detection limit of 0.2 nM, which is about 80 times lower than that from the bounded DNA signal based on 3 times of signal to noise. Good selectivity using water probe is also confirmed by the results from hybridization experiments with complementary DNA and mismatched DNA (Figure 4g).

CONCLUSION In summary, we propose a universal ATR-SEIRAS approach using the signal of the repelled water as an IR probe that can be successfully used to qualitatively and quantitatively monitor interfacial bioevents of different sized biomolecules (protein and DNA molecules, respectively) with high sensitivity, low detection limit and reliability in situ, lable free and real time. Using interfacial immuno-recognition and DNA hybridization as demonstrations, water as the IR probe offers 26 and 34 times higher sensitivity and even 200 and 86 times lower detection limit for immunosensing and DNA sensing, respectively, as compared to the traditional IR molecular fingerprints. With the help of specific recognition biointerfaces, the high selectivity of the developed method can be ensured. Further study would aim at taking advantages of water probe for application in bioanalysis and biomedical diagnosis and in the study of interfacial properties and water mediated events. 14


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation of China (21327902, 21635004). References (1) Osawa, M. Topics Appl. Phys. 2001, 81, 163. (2) Wang, H.; Zhou, Y.-W.; Cai, W.-B. Cur. Opin. Electrochem. 2017, 1, 73-79. (3) Fahrenfort, J. Spectrochim. Acta 1961, 17, 698-709. (4) Harrick, N. J. Phys. Rev. Lett. 1960, 4, 224-226. (5) Ataka, K.; Giess, F.; Knoll, W.; Naumann, R.; Haber-Pohlmeier, S.; Richter, B.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 16199-16206. (6) Ataka, K.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 9445-9457. (7) Grossutti, M.; Leitch, J.-J.; Seenath, R.; Karaskiewicz, M.; Lipkowski, J. Langmuir 2015, 31, 4411-4418. (8) Wang, G.-X.; Bao, W.-J.; Wang, M.; Xia, X.-H. Chem. Commun. 2012, 48, 1085910861. (9) Wang, G.-X.; Zhou, Y.; Wang, M.; Bao, W.-J.; Wang, K.; Xia, X.-H. Chem. Commun. 2015, 51, 689-692. (10) Ataka, K.; Heberle, J. Anal. Bioanal. Chem. 2007, 388, 47-54. (11) Ataka, K.; Kottke, T.; Heberle, J. Angew. Chem. Int. Ed. 2010, 49, 5416-5424. (12) Bao, W.-J.; Li, J.; Cao, T.-Y.; Li, J.; Xia, X.-H. Talanta 2018, 176, 124-129. (13) Bao, W.-J.; Yan, Z.-D.; Wang, M.; Zhao, Y.; Li, J.; Wang, K.; Xia, X.-H.; Wang, 15



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(32) Kaake, L. G.; Zou, Y.; Panzer, M. J.; Frisbie, C. D.; Zhu, X.-Y. J. Am. Chem. Soc. 2007, 129, 7824-7830. (33) Pohle, W.; Zhurkin, V. B.; Fritzsche, H. Biopolymers 1984, 23, 2603-2622. (34) Sukhorukov, B. I.; Semyeonov, M. A.; Maleev, V. Y.; Shabarchina, L. I. Biofizika+ 1979, 24, 611-619. (35) Tajmirriahi, H. A.; Naoui, M.; Ahmad, R. Biopolymers 1993, 33, 1819-1827. (36) Zhu, C.-F.; Zeng, Z.-Y.; Li, H.; Li, F.; Fan, C.-H.; Zhang, H. J. Am. Chem. Soc. 2013, 135, 5998−6001.

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203x194mm (150 x 150 DPI)


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Water as a Universal Infrared Probe for Bioanalysis in Aqueous

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