Vancomycin Determination by Disrupting Electron-Transfer in a

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Vancomycin Determination by Disrupting Electron-Transfer in a Fluorescence Turn-On Squaraine-Anthraquinone Triad Shue Mei Ng, Xiangyang Wu, M Faisal Khyasudeen, Pawel Jacek Nowakowski, Howe-Siang Tan, Bengang Xing, and Edwin K. L. Yeow ACS Sens., Just Accepted Manuscript • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Vancomycin Determination by Disrupting Electron-Transfer in a Fluorescence Turn-On Squaraine-Anthraquinone Triad

Shue Mei Ng,† Xiangyang Wu,† M. Faisal Khyasudeen, Paweł J. Nowakowski, Howe-Siang Tan, Bengang Xing, and Edwin K. L. Yeow*

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371

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ABSTRACT: A highly sensitive and selective probe for Vancomycin (Van) in aqueous and serum samples is developed in this study. The probe is based on a triad consisting of a near-infrared squaraine dye (Seta-640) conjugated to two anthraquinone molecules via Lys-D-Ala-D-Ala peptides. In the absence of Van, the close proximity and efficient electron-transfer from the excited Seta-640 dye to anthraquinone result in significant fluorescence quenching of the dye (“off”-state). When Van is added, the antibiotic molecules bind with high affinity to the -D-Ala-D-Ala ligands in a 2:1 stoichiometry (Van:triad); resulting in fluorescence recovery that is as high as 30-times (“on”state). Even though bound Van enhances the fluorescence by reducing the rate of (intrinsic) polarity-induced non-radiative decay process, this effect plays only a minor role. Instead, the main reason behind the observed fluorescence recovery after drug binding is the effective inhibition of electron-transfer; plausibly arising from a steric-induced lengthening of the spatial separation between electron donor and acceptor. The probe has detection limits of 7.0 and 96.9 nM in buffer and human serum, respectively, operates in the clinically relevant range, is insensitive to Van crystalline degradation product (CDP-1) and easy to operate by using a commonly available fluorescence spectrometer.

Keywords: vancomycin, electron transfer, squaraine dye, anthraquinone, molecular triad


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Vancomycin (Van) is a glycopeptide antibiotic that is used to treat patients afflicted with infections caused by penicillin-resistant Gram-positive bacteria, and is the drug of ´last resort´ for tackling the deadly methicillin-resistant Staphylococcus aureus (MRSA). In order to administer optimal dosages and to avoid ototoxicity/nephrotoxicity during systematic treatment of patients, therapeutic drug monitoring of Van is routinely performed in hospitals.1,2 Van determination in aquatic environment is also commonly conducted to ensure that elevated drug concentration is not present which will otherwise lead to adverse repercussions to the ecosystem.3 Therefore, the development of efficient and cost-effective methods to determine Van in both human serum and aqueous samples is necessary. Currently, the quantification of Van is achieved using several assays such as optical methods,4-6 high performance liquid chromatography (HPLC),7,8 ELISA-based methods9,10 and commercially available immunoassays (e.g., fluorescence polarization immunoassay, enzymemultiplied immunoassay, turbidimetric, etc.).11,12 Unfortunately, these assays suffer from limitations such as long operation time and low throughput (e.g., in HPLC), lack of standard operation protocols (e.g., in ELISA-based methods), cross-reactivity with Van crystalline degradation product (CDP-1) found in patients with renal failure (e.g., in immunoassays),13,14 and the need to use designated instruments from manufacturers (e.g., in commercial immunoassays). The mode of action of Van is the selective binding of the drug to the -D-Ala-D-Ala termini of the bacterial cell wall peptidoglycan via five hydrogen bonds (association constant Ka = 0.77 µM-1 in phosphate buffer)15 which leads to the inhibition of cell growth and eventual cell death.16,17 Recently, a sensor consisting of an array of silicon cantilevers functionalized with the peptide ligand Lys-D-Ala-D-Ala was introduced to probe Van.18,19 Upon interaction with the antibiotic, the cantilevers experience surface stress and deflection; giving rise to the detection of


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Van with 10 nM sensitivity. However, the design and implementation of a robust and reproducible way of immobilizing analyte-selective materials on cantilevers remain a major challenge that needs to be further addressed before cantilever-based sensors are employed as a standard assay for drug determination.20 In this work, a novel triad molecule composing of a near-infrared squaraine dye conjugated to two anthraquinone molecules via Lys-D-Ala-D-Ala peptides is used as a Van probe. Squaraine-based dyes have previously been utilized to detect biomolecules (e.g., proteins, nucleic acid),21-23 where the association between the two moieties changes the polarity of the environment around the dye and induces a fluorescence enhancement behaviour.24 In the case of our triad, significant fluorescence quenching occurs in both buffer solution and deproteinized human serum due to a charge-transfer reaction between the squaraine dye and conjugated anthraquinone; the latter being a good electron-acceptor. In the presence of Van, the drug binds to the two -D-Ala-D-Ala ligands which effectively reduces the emission quenching and restores fluorescence by as much as 30-times. The working mechanism of the triad probe is investigated using time-resolved spectroscopy which reveals that apart from altering the microenvironment polarity, the bound Van impedes electron-transfer efficiency, possibly by imposing a steric effect.

EXPERIMENTAL SECTION Details on the synthesis and characterization of the various compounds used in this study (Chart 1) are provided in the Supporting Information (SI).


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Calibration Curve in PBS. To obtain the calibration curve of triad 1 in PBS, appropriate amounts of the Van stock solutions were added to the triad to achieve the desired final Van concentrations. The sample was left to stand for three min before its absorbance and fluorescence were measured. The quantum yield (Ф) of triad 1 at each Van concentration was determined (see SI).

Calibration Curve in Human Serum (HS) Pre-treatment of HS. For tests in human serum (HS), the serum was first deproteinized using organic solvents before use. Methanol followed by acetonitrile were added to the thawed serum (1.25:1.25:1 v/v/v) and the mixture was vortexed for 10 min. The suspension was centrifuged (A23-6x100 rotor, SORVALL LYNX 6000 centrifuge, Thermo Scientific) at 12000 rpm for 10 min at 4 oC, and the supernatant was decanted and lyophilized. The dried powder was reconstituted in water, with a volume equal to the original volume of serum used. Any undissolved material was removed by ultracentrifugation (F23-48x1.5 rotor, SORVALL LYNX 6000 centrifuge, Thermo Scientific) at 23000 rpm for 10 min at 4 oC. The clear supernatant was left to stand in the dark at room temperature for 2 h before it was used in the serum tests. Calibration curve of triad 1 in HS. The procedure to obtain the calibration curve in HS is the same as described for PBS, except that the equilibration time was five min. The limit of detection (LOD) of triad 1 in both PBS and HS was determined from the equation LOD = 3σ/b, where σ is the standard deviation obtained from the fluorescence measurement of the triad in the absence of Van in PBS/HS and b is the slope derived after linearly fitting the calibration curves within certain ranges.


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Preparation of Van-spiked HS samples for recovery tests. The HS samples containing 9 or 3 µM of Van were separately prepared by spiking 5 mL of the untreated serum with an appropriate amount of the Van stock solution. The spiked samples were vortexed for 1 min and deproteinized in a similar way as described above, except that there is an additional washing step before the supernatant is lyophilized. This washing step was added to improve the recovery of Van by re-suspending the protein precipitates in a mixture of methanol, acetonitrile and water (1.25:1.25:1 v/v/v), and centrifuging the suspension at 12000 rpm for 10 min at 4 oC. The supernatant was decanted and added to the first batch of liquid. This was repeated for three times and the collected fractions of liquid were lyophilized. The % recovery is given by: B/A × 100%, where A is the added [Van] (i.e. 9 or 3 µM) and B is the concentration of Van determined from the calibration curve.

Steady-state Absorption and Fluorescence Spectroscopy. Steady-state absorption and fluorescence spectra were recorded on a UV/Vis spectrometer (Cary 100, Varian) and fluorescence spectrometer (Cary Eclipse, Varian), respectively. The dye concentrations used were ~ 1.4 µM. For fluorescence measurements, the samples were excited at 590 nm. Details on electrochemistry experiments are given in the SI.

Time-resolved Measurements. Time-resolved fluorescence spectroscopy measurements were carried out on a time-correlated single photon counting (TCSPC) spectrofluorimeter (FluoroCube, Horiba Jobin Yvon) with an excitation wavelength of 638 nm generated using a pulsed diode laser (NanoLED-635L, Horiba Jobin Yvon). The fluorescence lifetime decay profiles were collected at 650 nm and fitted using the Horiba Jobin Yvon Datastation software. The goodness


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of fit was evaluated from the reduced chi-square (χ2) value and the randomness of the weighted residuals. For the femtosecond-transient absorption (fs-TA) spectroscopy measurements, samples were dissolved in PBS to provide an OD of ~ 0.2 at ~ 640 nm in a 1 mm cell. The transient absorption spectra were collected using a set-up as described in the SI. All measurements were performed at room temperature and repeated two to three times to ensure reproducible results.

R3 R2 N R4

R7

-O S 3

R6 O

O-

SO3-

R1 N+ R5

O

N

O

R8

N+

O

O R

R

3: R = HN-Lys(AQ)- D-Ala-D-Ala-OH 1: R7 = R8 = HN-Lys(AQ)- D-Ala-D-Ala-OH 2: R7 = R8 = HN-Lys(AQ)- D-Ala-D-Lac-OH O O 4: R7 = HN-Lys(AQ)- D-Ala-D-Ala-OH, R8 = OH O 5: R7 = R8 = HN-Lys(Ac)- D-Ala-D-Ala-OH AQ: Ac: 6: R7 = HN-Lys(Ac)- D-Ala-D-Ala-OH, R8 = OH O Seta-640 di-carboxylic acid: R7 = R8 = OH (For R1-R6: Two of the R groups would bear the six-carbon linker for modification. The other R groups could be alkyl chains with or without sulphonate groups.)

Chart 1. Chemical structures of compounds used in this study. The exact structure of Seta-640 is not known.

RESULTS AND DISCUSSION Squaraine-Anthraquinone Triad vs. Cy5-Anthraquinone Triad. The triad 1, used in this study, consists of a squaraine dye (Seta-640)25 covalently attached to two Lys-D-Ala-D-Ala 7 ACS Paragon Plus Environment

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peptides (Chart 1). Each lysine (Lys) on the peptide is in turned conjugated to an anthraquinone. As will be discussed in the sections below, the quenched emission of triad 1 displays significant fluorescence recovery upon binding with Van in aqueous solutions (~ 30-times) and serum samples (~ 21-times). Furthermore, the fluorescence quantum yield of triad 1 in the presence of Van (32 µM) remains relatively constant over a period of 12 h, making it a highly stable sensor (Figure S1, SI). However, when Seta-640 dye in triad 1 is replaced with a near-infrared cyanine Cy5 dye (triad 3 in Chart 1), a weak fluorescence recovery takes place in the presence of Van in PBS (e.g., 3.6-times when concentration of Van, [Van] = 16.6 µM). When bound to Van, the absorption spectrum of triad 3 is broadened and its fluorescence intensity decreases with standing time (Figure S2, SI). Furthermore, the latter is accompanied by the precipitation of solid materials (Figure S2, SI); indicating the formation of insoluble aggregates when Van is bound to the triad containing Cy5. The exact reason for this observation is not clear at the moment, however, it can be concluded that triad 3 is unstable and does not perform satisfactorily as a Van probe. Therefore, unlike triad 1, triad 3 is not a suitable probe for Van, and the choice of the dye is important to ensure sensor stability and efficient fluorescence quenching and recovery in the absence and presence of the substrate, respectively.

Electron Transfer in Triad 1. The absorption and fluorescence spectra of triad 1 and reference compound 5 (i.e. Seta-640 dye conjugated to two Lys(Ac)-D-Ala-D-Ala peptides and does not contain anthraquinone, Chart 1) in PBS are shown in Figure S3 (SI) and 1A, respectively. Triad 1 absorbs and emits with peak maxima at λex = 641 nm and λem = 646 nm, respectively, both values are close to the reference compound 5 (λex = 638 nm and λem = 648 nm). The lack of a


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significant band shift or distortion of band shape indicates the absence of strong electronic couplings between the ground-states of the Seta-640 molecule and anthraquinone in triad 1.

Figure 1. (A) Fluorescence spectra of triad 1 and reference compound 5 in PBS. Excitation wavelength λex = 590 nm and concentrations of 1 and 5 are ~ 1.4 µM. Inset of (A) shows the fluorescence lifetime decay profiles of 1 and 5 in PBS (λex = 637 nm and monitoring emission at λem = 650 nm). (B) Kinetic profiles extracted at 481 nm of the fs-TA spectra of 1 and 5 (Figure S6, SI). Inset of (B) shows the kinetic profile of 1 for the first 5.2 ps.

Figure 1A shows that the fluorescence of triad 1 in PBS is significantly quenched when compared to reference compound 5. In particular, the fluorescence quantum yields (Φ) of triad 1 and reference compound 5 are 0.006 and 0.19, respectively. Clearly, the presence of the anthraquinone molecules in triad 1 causes the fluorescence quantum yield of the squaraine dye to be reduced by ca. 32-times. This is plausibly due to either an energy or charge-transfer process. Since there is no spectral overlap between the absorption spectrum of anthraquinone-2carboxylic acid and emission spectrum of reference compound 5 (Figure S4, SI), resonance


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energy transfer is not likely to be responsible for the observed fluorescence quenching in triad 1. On the other hand, the Gibbs free energy for electron-transfer from Seta-640 di-carboxylic acid (Chart 1) to anthraquinone-2-carboxylic acid was determined to be ∆Get = -0.941 eV; indicating that charge-transfer quenching is an energetically favorable process (Figure S5, SI). The fluorescence lifetime decay profiles of triad 1 and reference compound 5 in PBS, recorded using TCSPC, are given in the inset of Figure 1A. The bi-exponential lifetime decay of reference compound 5 yields lifetime components τ1 = 1290 ± 4 ps (contribution to the decay α1 = 67 %) and τ2 = 548 ± 9 ps (α2 = 33 %). On the other hand, the emission lifetime profile of triad 1 decays significantly faster and is described using a tri-exponential decay function with τ1 = 1081 ± 6 ps (α1 = 34 %), τ2 = 425 ± 8 ps (α2 = 50 %) and a short component τ3 = 100 ± 10 ps (α3 = 16 %). The latter is shorter than the resolution of the TCSPC instrument; suggesting that ultrafast quenching processes are present that are not detected by the TCSPC measurements. Femtosecond-transient absorption (fs-TA) measurements were conducted to examine the ultrafast quenching processes. Both reference compound 5 and triad 1 in PBS were pumped with a 640 nm laser pulse excitation and their fs-TA spectra recorded at various delay times (Figure S6, SI). The kinetic trace of reference compound 5, probed at 481 nm in the S1 excited-state absorption band, is described using a bi-exponential decay function with the long lifetime component fixed at τ1 = 1290 ps (α1 = 31 %) (from TCSPC) and a short lifetime component τ2 = 330 ± 10 ps (α2 = 69 %) (Figure 1B). Given that the maximum time-window of the fs-TA experiment is ~ 1 ns, the extracted τ2 component is comparable to that of the TCSPC result (i.e., 548 ps). τ1 and τ2 are therefore the lifetimes of the excited S1 state.26 Compared to reference compound 5, the kinetic trace of triad 1 at 481 nm decays at a significantly faster rate due to electron transfer from the excited S1 state of the Seta-640 dye to an anthraquinone (Figure 1B). 10 ACS Paragon Plus Environment

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The tri-exponential decay fit to the kinetic trace yields lifetime components τ1 = 230 ± 30 ps (α1 = 6 %), τ2 = 2.9 ± 0.3 ps (α2 = 29 %) and τ3 = 0.32 ± 0.01 ps (α3 = 65 %). Results from both the TCSPC and fs-TA measurements indicate the existence of a wide range of electron-transfer rates in triad 1. This is because the electron donor and acceptor, in a fluid aqueous environment, are able to adopt several spatial conformations with respect to each other due to the flexible linker. The Marcus rate transfer for non-adiabatic charge-transfer is expressed as:27  2 π  k ET = h −1 V  k T λ B  

0.5

2   exp −  ∆G o + λ  / 4λk BT     

where V is the electronic coupling between donor and acceptor separated by a distance r, ∆Go is the free energy change for charge-transfer and λ is the total reorganization energy. When the Seta-640 dye is within close proximity to anthraquinone (i.e., short r), the electron-transfer reaction is highly efficient which results in the ultrafast quenching processes. Since V decreases exponentially with increasing r, at longer donor-acceptor spatial separations (i.e., long r), the electron-transfer rate becomes significantly slower. It is interesting to note that for triad 1, the ~ 1 ns lifetime component observed in the TCSPC measurement due to structures with long donoracceptor distances are not present in the fs-TA kinetic trace. This is possibly due to a small population of triad 1 with such conformations; in agreement with the low fluorescence quantum yield observed.


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Figure 2. Fluorescence spectra of (A) reference compound 5 and (C) triad 1 in the presence of different concentrations of Van in PBS (5: [Van] = 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 8, 16, 32 µM; 1: [Van] = 0, 0.5, 0.7, 1, 1.4, 1.7, 2, 2.3, 2.7, 3, 3.5, 4, 6, 8, 16, 32 µM). Inset shows the plot of Ф/Ф0 against [Van] and the black line in (C) shows the linear fit for the range of [Van] from 0 to 4 µM. The concentrations of 1 and 5 are ~ 1.4 µM. (B) Fluorescence lifetime decay profiles of triad 1 and reference compound 5 in the presence of 32 µM of Van in PBS (λex = 637 nm and monitoring emission at 650 nm). (D) Fluorescence spectra of triad 1 (~ 1.4 µM) in the presence of different concentrations of Van in HS ([Van] = 0, 2, 4, 6, 8, 10, 12, 16, 32 µM). Inset shows the plot of Ф/Ф0 vs. [Van] and the black line shows the linear fit for the range of [Van] from 0 to 12 µM.


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Vancomycin Inhibits Electron Transfer in Triad 1. Upon the addition of Van to reference compound 5 in PBS, the absorption spectrum is slightly red-shifted as illustrated in Figure S7 (SI) (e.g., when [Van] = 32 µM, λabs is red-shifted by 3 nm). The fluorescence of reference compound 5 is enhanced in the presence of increasing concentrations of Van (Figure 2A). In particular, the emission quantum yield is increased from Φ0 = 0.19 in the absence of Van to Φ = 0.35 when [Van] = 32 µM (i.e., 1.8-times increase) (see the plot of Φ/Φ0 vs. [Van] in the inset of Figure 2A, where Φ are the quantum yields in the presence of different Van concentrations). The binding stoichiometry between reference compound 5 and Van was determined using the mole ratio method to be 1:2 (Figure S8A, SI). Assuming that the formation of the 1:2 complex follows a stepwise equilibrium reaction between 5 and Van, the association constant for the formation of the 1:1 complex (5 + Van from 5:Van + Van

→ ←

→ ←

5:Van) and stepwise association constant of 5:(Van)2

5:(Van)2 were determined to be K11 = 0.75 µM-1 and K12 = 0.51 µM-1,

respectively (Figure S8B, SI). The value of K11 is in agreement with previously reported binding constant between Van and -D-Ala-D-Ala ligand.15 If the initial concentration of 5 is 1.4 µM, then at 2 (21.4) equivalents of Van, the proportions of 5 in the form of 5:(Van)2, 5:(Van) and unbound 5 in solution are 28.4 % (93.0 %), 37.6 % (6.7 %) and 34.0 % (0.3 %), respectively. Clearly, at 21.4 equivalents (or 30 µM) of Van, the solution contains predominantly 1:2 complex. The TCSPC fluorescence lifetime profile of reference compound 5 in the presence of 32 µM of Van in PBS is fitted to a bi-exponential decay function with lifetime components τ1 = 2290 ± 5 ps (α1 = 74 %) and τ2 = 980 ± 20 ps (α2 = 26 %) (Figure 2B). The corresponding averaged lifetime = 2121 ps, calculated from = ∑ i α iτ i2 / ∑ i α iτ i , is longer than = 1162 ps when [Van] = 0 by 1.8-times (inset of Figure 1A); in agreement with the increase in


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fluorescence quantum yield. The averaged fluorescence () and non-radiative () decay constants are computed from: = Φ/ = (Φ-1 – 1) In the absence of Van, = 0.16 ns-1 and = 0.70 ns-1, whereas for [Van] = 32 µM, = 0.17 ns-1 and = 0.31 ns-1. Upon binding to Van via the two -D-Ala-D-Ala ligands, the Seta640 dye in reference compound 5 comes into close proximity to the drug molecules, leading to a reduction in the polarity of the surrounding environment. The change in polarity alters more significantly than .24 In this case, the polarity-induced non-radiative process is less efficient in a more non-polar environment which leads to an enhancement in the fluorescence quantum yield of the dye when bound to Van (Figure 2A).24 A control experiment was conducted whereby 32 µM of Van is added to Seta-640 di-carboxylic acid in PBS (i.e., dye not conjugated to Lys(Ac)-D-Ala-D-Ala) and an insignificant change in the emission quantum yield is observed (Figure S9, SI). For triad 1 in PBS, the addition of Van causes the fluorescence intensity and quantum yield to increase significantly (Figure 2C). For example, Φ0 = 0.006 in the absence of Van is increased to Φ = 0.18 when [Van] = 32 µM (i.e., ca. 30-times enhancement). The influence of bound Van on the intrinsic polarity-induced non-radiative decay (i.e., 1.8-times increase in Φ) is unable to completely explain the increase in fluorescence quantum yield observed in Figure 2C. We propose that the reduction in polarity due to the bound Van may reduce the electron-transfer efficiency in triad 1. In addition, when Van molecules bind to the -D-Ala-D-Ala ligands in triad 1, the anthraquinones are likely to be pulled away from the Seta-640 dye due to steric effect (Scheme 1). The increase in spatial separation between the electron donor and acceptor in the 14 ACS Paragon Plus Environment

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presence of Van reduces the charge-transfer quenching efficiency and results in the observed enhancement in fluorescence intensity.

Scheme 1. Proposed mechanism of fluorescence recovery of triad 1 in the presence of Van. In the absence of Van, the anthraquinone molecules are in close proximity to the squaraine dye and electron-transfer (ET) from the photo-excited dye to anthraquinone occurs. When the triad binds to Van via -D-Ala-D-Ala ligands, the anthraquinone molecules are positioned further away from the dye; possibly due to steric hindrance. This results in the decrease of ET efficiency and fluorescence recovery occurs.

The fluorescence recovery of triad 1 reaches a plateau at ca. [Van] = 30 µM (Figure 2C) with an emission quantum yield (Φ = 0.18) that is lower than that of reference compound 5 in the presence of the same amount of drug (Φ = 0.35). In order to gain further insight into this observation, time-resolved experiments were conducted. In the case of triad 1 in the presence of 32 µM of Van in PBS, the fluorescence lifetime profile from TCSPC shows a bi-exponential decay with components τ1 = 1975 ± 3 ps (α1 = 90 %) and τ2 = 740 ± 30 ps (α2 = 10 %) (Figure 15 ACS Paragon Plus Environment

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2B). The averaged lifetime = 1926 ps is close in value to that of reference compound 5 in the presence of the same amount of antibiotic (i.e., 2121 ps), and arises from triad conformers that do not undergo electron-transfer quenching due to the long electron donor-acceptor separation after binding with Van. However, ultrafast quenching processes are still present in triad 1 as revealed by fs-TA measurements (Figure S10, SI); arising from a portion of Van-1-Van triads whose electron donor-acceptor distances are still sufficiently short to induce rapid electron-transfer reactions. The fluorescence recovery experiment by Van was also conducted on triad 2 (Chart 1), consisting of a Seta-640 dye covalently attached to two Lys-D-Ala-D-Lac peptides. Each Lys on the peptide is in turned conjugated to an anthraquinone. In this case, the quenched fluorescence quantum yield of triad 2 in PBS is Φ0 = 0.005; similar to the quantum yield of triad 1. Upon the addition of 50 µM of Van, the fluorescence quantum yield increased only slightly to Φ = 0.006 (Figure S11, SI). The loss of a hydrogen bond and a gain of an oxygen-oxygen lone pair repulsion between -D-Ala-D-Lac and Van have been reported to give rise to a 1000-fold reduction in affinity between the antibiotic and peptide ligand as compared to -D-Ala-D-Ala.15 Therefore, the lack of fluorescence recovery for triad 2 is due to the significantly weaker binding between Van and -D-Ala-D-Lac ligand, and supports the existence of specific binding between Van and -D-Ala-D-Ala ligand in triad 1. The specific binding interaction between Van and triad 1, and subsequent inhibition of electron-transfer quenching and fluorescence recovery are the driving mechanisms behind the application of triad 1 in the detection and quantification of Van in aqueous solution. The limit of detection (LOD) of triad 1 in PBS was determined to be 7.0 nM (inset of Figure 2C) which is lower than the LOD of chemiluminescence-based methods discussed elsewhere,28 and is


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comparable to assays using ultra performance liquid chromatography-tandem mass spectrometry.29 A dyad 4 containing a Seta-640 dye conjugated to an anthraquinone via a Lys-D-Ala-DAla peptide (Chart 1) displays a quenched fluorescence quantum yield (Φ = 0.007 in PBS) that increases by only 2.9-times when 32 µM of Van was added into the solution (Figure S12, SI). The poor fluorescence recovery in dyad 4 compared to triad 1 implies a smaller degree of electron-transfer inhibition in the dyad upon binding to Van. This could be due to a weaker steric hindrance effect in dyad 4 arising from one bound Van as compared to two bound Vans in triad 1 which allows the flexibly linked anthraquinone to remain close to the dye in the dyad.

Quantification of Van in Human Serum. Before determination of Van in human serum (HS), the serum is first pre-treated with organic solvents to remove proteins30,31 (e.g., albumin and immunoglobulin A) which can bind with either Van or squaraine.21,32,33 Figure 2D shows the fluorescence recovery of triad 1 upon addition of increasing amounts of Van. It is observed that the emission quantum yield of the triad in the absence of Van (Φ = 0.003) is increased by 21times when 32 µM of Van was added. The maximum recovered fluorescence intensity in the presence of 32 µM of Van is lower for HS as compared to PBS (i.e., 30-times in PBS). A plausible explanation for the discrepancy is the presence of trace amounts of proteins remaining in the serum after the protein precipitation process or endogenous non-protein compounds which bind to Van or squaraine. This decreases the concentrations of free Van or unbound triad, and may also lead to a disruption in the triad-Van association. Nevertheless, the working range of triad 1 covers the clinically therapeutic window of Van (i.e., 7 - 13.5 µM),34 and is still able to recover the concentration of Van in HS with relatively high accuracy as discussed next.


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HS samples spiked with two concentrations of Van (9 and 3 µM) were deproteinized, and the fluorescence spectra of the final solutions containing triad 1 recorded. The [Van] values obtained from the calibration curve indicate a Van recovery within ± 20 % of the actual value (recovered [Van] = 9.07 and 2.38 µM) which is comparable to other Van determination assays.8 In addition, triad 1 detects Van with high sensitivity in HS. A LOD of 96.9 nM is obtained from fitting the linear range of the calibration curve (inset of Figure 2D) which is lower than the LODs of assays using high performance liquid chromatography coupled with various detection methods.35-37

Selectivity and Interference Tests. To investigate whether triad 1 responds to other commonly used antibiotics, 100 µM of Tobramycin, Gentamicin, Amikacin and Penicillin G were separately added to a PBS solution of the probe. From Figure 3A, it is clearly seen that while the fluorescence quantum yield of triad 1 is enhanced in the presence of Van (i.e., Ф/Φ0 >> 1), it remains relatively invariant (i.e., Ф/Φ0 ~ 1) when an excess of the other antibiotics was utilized. This demonstrates that triad 1 does not respond to Tobramycin, Gentamicin, Amikacin and Penicillin G, and is selective towards Van. The interference of some common coexisting substances on the detection of Van was also examined by adding glycine, L-alanine, urea, D-glucose, ascorbic acid, Mg2+, SO42- and CO32separately to a PBS solution of triad 1 in the presence of Van (2 µM). The coexisting substance is considered to have an insignificant interference if the fluorescence intensity of the sample is within ± 5% of the intensity of the control sample (i.e., triad 1 with 2 µM Van only). It is observed in Figure 3B that the addition of the coexisting substances does not perturb the fluorescence quantum yield of triad 1 in the presence of Van.


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Figure 3. (A) Selectivity of triad 1. Different antibiotics (32 or 100 µM) were added to an aqueous solution of triad 1 (~ 1.4 µM) in the absence of Van in PBS (Tob: Tobramycin, Gen: Gentamicin, Ami: Amikacin, Pen G: Penicillin G, CDP-1: Van CDP-1 (32 µM), Van (32 µM), Triad 1). The sample with triad 1 only was used as the control. (B) Interference test. 0.1 or 2 mM of common co-existing substances were added to an aqueous solution of triad 1 (~ 1.4 µM) in the presence of 2 µM of Van in PBS (Gly: Glycine, L-Ala: L-Alanine, D-Glu: D-Glucose, Mg2+: MgCl2·6H2O, SO42-: Na2SO4, CO32-: Na2CO3 (0.1 mM), AA: Ascorbic acid (0.1 mM)). The sample with triad 1 and 2 µM of Van was used as the control.

A common problem encountered in immunoassays of Van determination is the crossreactivity of Van crystalline degradation product (CDP-1) which gives false positive results.38,39 Harris et al. have proposed that an additional methylene group in the peptide backbone of Van CDP-1 causes it to assume a conformation that does not facilitate efficient binding with small aliphatic peptides such as Ac-D-Ala-D-Ala; rendering Van CDP-1 inactive against Gram-positive bacteria.40 When Van CDP-1 (32 µM) was added to a PBS solution of triad 1, no significant change in the fluorescence quantum yield is observed (Figure 3A). An obvious advantage of 19 ACS Paragon Plus Environment

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triad 1 over immunoassays is therefore its ability to distinguish between Van and its crystalline degradation product with high selectivity towards the former.

CONCLUSION A highly sensitive and selective probe for Van is developed in this study that quantifies the drug in both aqueous and serum samples. In the absence of Van, photo-induced electron-transfer from the excited Seta-640 dye to anthraquinone in triad 1 results in significant fluorescence quenching of the dye. When Van is added, the antibiotic molecules bind with high affinity to the -D-Ala-DAla ligands, resulting in fluorescence recovery that can be as high as 30-times. Even though bound Van enhances the fluorescence by reducing the rate of the (intrinsic) polarity-induced non-radiative decay process, this effect plays only a minor role. Instead, the main reason behind the observed fluorescence recovery after drug binding is the effective inhibition of electrontransfer; plausibly arising from a reduction in environmental polarity and steric-induced lengthening of the spatial separation between squaraine dye and anthraquinone. The proposed Van probe is insensitive to other antibiotics and Van CDP-1, operates in the clinically relevant range, and is easy to operate by using a commonly available fluorescence spectrometer.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: The following material is available free of charge via the Internet at http://pubs.acs.org.


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Details on the synthesis and characterization of compounds, fs-transient absorption (TA) spectroscopy setup, and experimental data on electrochemistry, fs-TA spectroscopy, binding stoichiometry are provided. AUTHOR INFORMATION Corresponding Author Email: [email protected] Author Contributions S.M.N. and X.W. contributed equally to the work. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT Financial support from the Singapore Ministry of Education MOE Tier 1 grants (RG6/15) and (RG16/15) are acknowledged. REFERENCES (1)

Hammett-Stabler, C. A.; Johns T. Laboratory guidelines for monitoring of antimicrobial

drugs. Clin. Chem. 1998, 44, 1129-1140. (2)

Finch, R. G.; Elipoulos, G. M. Safety and efficacy of glycopeptide antibiotics. J.

Antimicrob. Chemother. 2005, Suppl. S2, ii5-ii13. (3)

Kümmerer, K. Antibiotics in the aquatic environment – a review – part 1. Chemosphere

2009, 75, 417-434. (4)

Yu, L.; Zhong, M.; Wei, Y. Direct fluorescence polarization assay for the detection of

glycopeptide antibiotics. Anal. Chem. 2010, 82, 7044-7048.


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(5)

Page 22 of 27

Liang, W.; Liu, S.; Liu, Z.; Li, D.; Wang, L.; Hao, C.; He, Y. Electron transfer and

fluorescence “turn-off” based CdTe quantum dots for vancomycin detection at nanogram level in aqueous serum media. New. J. Chem. 2015, 39, 4774-4782. (6)

Korposh S.; Chianella, I.; Guerreiro, A.; Caygill, S.; Piletsky, S.; James, S. W.; Tatam, R.

P. Selective vancomycin detection using optical fibre long period gratings functionalized with molecularly imprinted polymer nanoparticles. Analyst 2014, 139, 2229-2236. (7)

Pfaller, M. A.; Krogstad, D. J.; Granich, G. G.; Murray, P. R. Laboratory evaluation of

five assays for vancomycin: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay, radioimmunoassay, and fluorescence immunoassay. J. Clin. Mirobiol. 1984, 20, 311-316. (8)

Javorska, L.; Krcmova, L. K.; Solichova, D.; Solich, P.; Kaska, M. Modern methods for

vancomycin determination in biological fluids by methods based on high-performance liquid chromatography - a review. J. Sep. Sci. 2016, 39, 6-20. (9)

Odekerken, J. C. E.; Logister, D. M. W.; Assabre, A.; Arts, J. J. C.; Walenkamp, G. H. I.

M.; Welting, T. J. M. ELISA-based detection of gentamicin and vancomycin in proteincontaining samples. SpringerPlus 2015, 4, 614. (10)

Chianella, I.; Guerreiro, A.; Moczko, E.; Caygill, J. S.; Piletska, E. V.; Perez De Vargas

Sansalvador, I. M.; Whitcombe, M. J.; Piletsky, S. A. Direct replacement of antibodies with molecularly imprinted polymer nanoparticles in ELISA - development of a novel assay for vancomycin. Anal. Chem. 2013, 85, 8462-8468. (11)

White, L. O.; Holt, H. A.; Reeves, D. S., MacGowan, A. P. Evaluation of innofluor

fluorescence polarization immunoassay kits for the detection of serum concentrations of


Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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gentamicin, tobramycin, amikacin and vancomycin. J. Antimicrob. Chemother. 1997, 39, 355361. (12)

Yeo, K. –T.; Traverse, W.; Horowitz, G. L. Clinical performance of the EMIT

vancomycin assay. Clin. Chem. 1989, 35, 1504-1507. (13)

Wilson, J. F.; Davis, A. C.; Tobin, C. M. Evaluation of commercial assays for

vancomycin and aminoglycosides in serum: a comparison of accuracy and precision based on external quality assessment. J. Antimicrob. Chemother. 2003, 52, 78-82. (14)

Anne, L.; Hu, M.; Chan, K.; Colin, L.; Gottwald, K. Ther. Drug. Monit. Potential

problem with fluorescence polarization immunoassay cross-reactivity to vancomycin degradation product CDP-1: its detection in sera of anally impaired patients. 1989, 11, 585-591. (15)

Xing, B.; Jiang, T.; Wu, X.; Liew, R.; Zhou, J.; Zhang, D.; Yeow, E. K. L. Molecular

interactions between glycopeptide vancomycin and bacterial cell wall peptide analogues. Chem. Eur. J. 2011, 17, 14170-14177. (16)

Xing, B.; Yu, C. –W.; Ho, P. –L.; Chow, K. –H.; Cheung, T.; Gu. H.; Cai, Z.; Xu, B.

Multivalent antibiotics via metal complexes: potent divalent vancomycins against vancomycinresistant enterococci. J. Med. Chem. 2003, 46, 4904-4909. (17)

Xing, B.; Jiang, T.; Bi, W.; Yang, Y.; Li, L.; Ma, M.; Chang, C. –K.; Xu, B.; Yeow, E. K.

L. Multifunctional divalent vancomycin: the fluorescent imaging and photodynamic antimicrobial properties for drug resistant bacteria. Chem. Commun. 2011, 47, 1601-1603. (18)

Ndieyira, J. W.; Watari, M.; Barrera, A. D.; Zhou, D.; Vögtl, M.; Batchelor, M.; Cooper,

M. A.; Strunz, T.; Horton, M. A.; Abell, C.; Rayment, T.; Aeppli, G.; McKendry, R. A. Nanomechanical detection of antibiotic-mucopeptide binding in a model for superbug drug resistance. Nat. Nanotechnol. 2008, 3, 691-696.


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(19)

Page 24 of 27

Ndieyira, J. W.; Kappeler, N.; Logan, S.; Cooper, M. A.; Abell, C.; McKendry, R. A.;

Aeppli, G. Surface-stress sensors for rapid and ultrasensitive detection of active free drugs in human serum. Nat. Nanotechnol. 2014, 9, 225-232. (20)

Boise, A.; Thundat, T. Design and fabrication of cantilever array biosensors. Mater.

Today 2009, 12, 32-38. (21)

Terpetschnig, E.; Szmacinski, H.; Ozinskas, A.; Lakowicz, J. R. Synthesis of squaraine-

N-hydroxysuccinimide esters and their biological application as long-wavelength fluorescent labels. Anal. Biochem. 1994, 217, 197-204. (22)

Sleiman, M. H.; Ladame, S. Synthesis of squaraine dyes under mild conditions:

applications for labelling and sensing of biomolecules. Chem. Commun. 2014, 50, 5288-5290. (23)

Xia, G.; Wang, H. Squaraine dyes: the hierarchical synthesis and its application in optical

detection. J. Photochem. Photobiol. C 2017, 31, 84-113. (24)

Cornelissen-Gude, C.; Rettig, W.; Lapouyade, R. Photophysical properties of squaraine

derivatives: evidence for charge separation. J. Phys. Chem. A 1997, 101, 9673-9677. (25)

Patsenker, L.; Tatarets, A.; Kolosova, O.; Obukhova, O.; Povrozin, Y.; Fedyunyayeva, I.;

Yermolenko, I.; Terpetschnig, E. Fluorescent probes and labels for biomedical applications. Ann. N. Y. Acad. Sci. 2008, 1130, 179-187. (26)

de Miguel, G.; Marchena, M.; Zitnan, M.; Pandey, S. S.; Hayase, S.; Douhal, A. Femto to

millisecond observations of indole-based squaraine molecules photodynamics in solution. Phys. Chem. Chem. Phys. 2012, 12, 1796-1805. (27)

Wu, X.; Liu, F.; Wells, K. L.; Tan, S. L. J.; Webster, R. D.; Tan, H. –S.; Zhang, D.; Xing,

B.; Yeow, E. K. L. Interplay of hole transfer and host-guest interaction in a molecular dyad and


Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

triad: ensemble and single-molecule spectroscopy and sensing applications. Chem. Eur. J. 2015, 21, 3387-3398. (28)

Khataee, A. R.; Hasanzadeh, A.; Iranifam, M., Fathinia, M.; Hanifehpour, Y.; Joo, S. W.

CuO nanosheets-enhanced flow-injection chemiluminescence system for determination of vancomycin in water, pharmaceutical and human serum. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014, 122, 737-743. (29)

Li, B.; Zhang, T.; Xu, Z.; Fang, H. H. P. Rapid analysis of 21 antibiotics of multiple

classes in municipal wastewater using ultra performance liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta 2009, 645, 64-72. (30)

Muppidi, K.; Pumerantz, A. S.; Betageri, G.; Wang, J. Development and validation of a

rapid high-performance liquid chromatography method with UV detection for the determination of vancomycin in mouse plasma. J. Chromat. Separation Techniq. 2013, 4, 1-5. (31)

Tariq, M. H.; Naureen, H.; Abbas, N.; Akhlaq, M. Development and validation of a

simple, accurate and economical method for the analysis of vancomycin in human serum using ultracentrifuge protein precipitation and UV spectrophotometer. J. Anal. Bioanal. Tech. 2015, 6, 1-5. (32)

Sun. H.; Maderazo, E. G.; Krusell, A. R. Serum protein-binding characteristics of

vancomycin. Antimicrob. Agents Chemother. 1993, 37, 1132-1136. (33)

Butterfield, J. M.; Patel, N.; Pai, M. P.; Rosano, T. G.; Drusano, G. L.; Lodise, T. P.

Refining vancomycin protein binding estimates: identification of clinical factors that influence protein binding. Antimicrob. Agents Chemother. 2011, 55, 4277-4282. (34)

Rybak, M. J.; Lomaestro, B. M.; Rotschafer, J. C.; Moellering, Jr., R. C.; Craig, W. A.;

Billeter, M.; Dalovisio, J. R.; Levine, D. P. Vancomycin therapeutic guidelines: a summary of


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

consensus recommendations from the infectious diseases society of America, the American society of health-system pharmacists, and the society of infectious diseases pharmacists. Clin. Infect. Dis. 2009, 49, 325-327. (35)

Lopez, K. J. V.; Bertoluci D. F.; Vicente, K. M.; Dell’Aquilla, A. M.; Santos, S. R. C. J.

Simultaneous determination of cefepime, vancomycin and imipenem in human plasma of burn patients by high-performance liquid chromatography. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007, 860, 241-245. (36)

Hagihara, M. Sutherland, C.; Nicolau, D. Development of HPLC methods for the

determination of vancomycin in human plasma, mouse serum and bronchoalveolar lavage fluid. J. Chromatogr. Sci. 2013, 51, 201-207. (37)

Favetta, P.; Guitton, J.; Bleyzac, N.; Dufresne, C.; Bureau, J. New sensitive assay of

vancomycin

in

human

plasma

using

high-performance

liquid

chromatography

and

electrochemical detection. J. Chromatogr. B 2001, 751, 377-382. (38)

Tanaka, M.; Orii, T.; Gomi, T.; Kobayashi, H.; Kanke, M.; Hirono, S. Clinical estimation

of vancomycin measurement method on hemodialysis patient. Yakugaku Zasshi 2002, 122, 269275. (39)

Somerville, A. L.; Wright, D. H., Rotschafer, J. C. Implications of vancomycin

degradation products on therapeutic drug monitoring in patients with end-stage renal disease. Pharmacotherapy 1999, 19, 702-707. (40)

Harris, C. M.; Kopecka, H.; Harris, T. M. Vancomycin: structure and transformation to

CDP-1. J. Am. Chem. Soc. 1983, 105, 6915-6922.


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TOC:


Vancomycin Determination by Disrupting Electron-Transfer in a

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