Design of controlled multi-probe coupled assay via bioinspired signal amplification approach for mercury detection

Abstract

We have designed and synthesized off–on fluorometric probes, 7-(but-3-yn-1-yloxy)-8-(difluoromethyl)-2H-chromen-2-one (AYF) and 7-((4-(tert-butyldiphenylsilyloxy)benzyl)oxy)-8-(difluoromethyl)coumarin (DPF₁), by incorporating the concept of biological signal transduction strategy into their molecular design and present their proof-of-concept demonstration for mercury (Hg²⁺) detection. AYF undergoes a cascade of self-immolative reactions in the presence of Hg²⁺ and unmasks fluorogenic coumarin 1 and releases two latent fluoride ions. These fluorides initiate the removal of silyl protecting trigger group on DPF₁ which directs another cascade of self-immolative reactions. The fluoride ions (amplifiers) are continuously activating the cascade reaction, and, as a result, coumarin 1 progressively accumulates in the reaction medium and the whole process accelerates an amplified signal for Hg²⁺. For detection of Hg²⁺, the limit of detection of the two-probe coupling assay (AYF and DPF₁) is 0.51 ppb which is more than 2000 times lower than that of AYF alone. Besides, 3-(benzothiazol-2-yl)-4-carbonitrile-7-((4-(tert-butyldiphenylsilyloxy)benzyl)oxy)coumarin (DCC), a long-wavelength probe, was coupled with AYF and DPF₁ in order to shift the emission to a higher wavelength region. Negative control studies revealed that the probes undergo controlled and organized sensing pathways as we designed.

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1. Introduction

The development of novel sensing approaches and methods in order to improve sensitivity and selectivity of analytical methods is an active area of research in analytical chemistry.¹ A sensing method seeks to change unknown information (identity, quality, reactivity, etc.) in a chemical or biological sample into measurable signals. The signals can be obtained from various transduction techniques, where the unknown information from the sample is then transformed into optical,^2,3 electrical,^4,5 or mechanical⁶ readouts. The currently known sensitive and selective techniques such as enzyme-linked immunosorbent assay (ELISA),⁷ polymerase chain reaction (PCR),⁸ and bio-barcode assays⁹ usually rely on antibodies, enzymes or biomolecules¹⁰ to achieve high sensitivity and selectivity. However, in most cases these reagents lack thermal stability and are not stable for prolonged periods; as a result they encounter practical difficulties in performing real-application analysis at ambient temperature. In order to circumvent these limitations, recently several research groups have reported the design of auto-inductive molecular probes by incorporating the signal amplification concept of biomolecules to achieve high sensitivity and selectivity.^11–16 Biological systems recognize and respond to external signals or stimulants through signal transduction processes, where the original signal is amplified into other chemical information through well-organized catalytic processes.¹⁷ Generally, the auto-inductive molecular probes are equipped with unique triggering groups which selectively interact with the analyte of interest via a designated chemical reactions pathway. The analyte induces the designated chemical reactions which leads to a cascade of self-immolative reactions and unmasks the reporter molecules (chromogenic or fluorogenic) and spontaneously liberates the signal transduction molecules. These signal transduction molecules continuously induce the auto-inducible molecular probes to undergo self-immolative signal-revealing reactions which accumulate reporter molecules, and this cyclic process facilitates amplification (Scheme 1).

Scheme 1 A general scheme for signal amplification strategy.

Here, we sought to design auto-inductive fluorescent probes by incorporating the biological signal transduction concept and demonstrate their proof-of-concept for mercury ion (Hg²⁺) detection. Hg²⁺ is one of the most toxic and highly poisonous metal ions and a widespread pollutant of natural and anthropogenic sources. Organic forms of mercury, such as methylmercury species, are much more toxic than inorganic mercury species.¹⁸ Notably, methylmercury species readily cross the blood–brain barrier due to their lipid solubility and accumulate in the brain which can cause damage to the central nervous system and other organs as well.^19,20 Thus, the active monitoring of Hg²⁺ in our environment is needed to ensure proper physiological function and food safety,²¹ and the development of novel approaches for the sensitive detection of Hg²⁺ is extremely important.^22,23

Designing latent self-immolative ratiometric chemical probes for sensor applications is of ongoing interest in our research group.^24–26 Recently, we have successfully designed an auto-inducible off–on fluorogenic ratiometric probe for ultrasensitive fluoride detection.²⁵ We are interested in expanding the similar signal-revealing mechanism to design a new bioinspired sensing approach for Hg²⁺ detection using the auto-inductive off–on fluorogenic probes AYF and DPF₁ (Scheme 2). The probe AYF is composed of a 3-butynyl group attached via ether linkage to the latent reporter coumarin which carries two fluorides, and these fluorides act as signal amplifiers. The Hg²⁺ facilitates the removal of the 3-butynyl group from AYF via oxymercuration reaction which results in a cascade of self-immolative reactions through quinone-methide type of rearrangement to generate fluorogenic reporter 8-formyl-7-hydroxycoumarin (coumarin 1),^27,28 with concomitant release of two fluorides.²⁹ Notably, Koide et al. successfully demonstrated the Hg²⁺-promoted cleavage of fluorescence-masking 3-butyne or vinyl groups in low ppb ranges under mild condition.^27,28 The latent generated fluorides then induce the removal of silyl protecting trigger group on DPF₁ which results in another cascade of self-immolative reactions in a similar way as AYF and eject coumarin 1 and two additional fluorides. The released fluorides are highly capable of continuously inducing the self-immolative reactions of unreacted DPF₁, and this cyclic process leads to the accumulation of fluorogenic coumarin 1 which results in exponential signal amplification. The main objective of this work is to design a controlled multi-probe coupling assay incorporating the concept of biological signal transduction for Hg²⁺ detection and demonstrate its proof-of-concept for Hg²⁺. To our knowledge, this is the first report which incorporates the biological signal amplification concept for Hg²⁺ detection.

Scheme 2 Pathway by which AYF detects Hg²⁺ and releases two equivalents of fluorides to propagate the auto-inductive signal amplification process.

2. Experimental

2.1. Chemicals and instrumentation

The chemicals were purchased from Acros Organics, Sigma-Aldrich, Showa Chemical Industry Co., or TCI America and were used without further purification. All reactions requiring anhydrous conditions were performed in oven-dried glassware under an Ar or N₂ atmosphere. Chemicals and solvents were either puriss p.a. or purified by standard techniques. Analytical thin-layer chromatography was performed using glass plate-mounted silica gel 60F₂₅₄ (Merck) with a thickness of 0.2 mm. Flash column chromatography was performed using Silicycle silica gel 60. The synthesized compounds were characterized by using ¹H NMR (Bruker Advance 300 MHz) and IR spectroscopy (HORIBA 720 infrared spectrophotometer). Mass spectra were recorded using a Finnigan TSQ 700 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source. Photoluminescence spectra were measured with a HORIBA FluoroMax-4 spectrometer. 0.1 M citric acid BIS-TRIS propane buffer (CBTP) (pH 7.1 and 7.6) was prepared with CBTP in water.

2.2. Synthesis of 7-(but-3-yn-1-yloxy)-2-oxo-2H-chromene-8-carbaldehyde (2)

A mixture of 7-hydroxy-8-aldehydecoumarin (1, 0.05 g, 0.263 mmol) and potassium carbonate (K₂CO₃, 0.109 g, 0.788 mmol) was dissolved in 1.314 mL anhydrous N,N-dimethylformamide (DMF) with subsequent addition of 3-butynyl p-toluenesulfonate (0.175 mL, 0.788 mmol) to the mixture. The whole mixture was stirred under a stream of argon (Ar) at 84 °C for 24 h and the reaction mixture was extracted with dichloromethane (DCM)/water. The organic layer was dried over anhydrous magnesium sulfate (MgSO₄). The solvent was removed and the residue was purified by column chromatography on silica gel (eluent: ethyl acetate/n-hexane = 1 : 1) yielding the title compound 2 as a white solid (78%, 0.0492 g). ¹H NMR (CDCl₃, 300 MHz, ppm): δ = 10.63 (s, 1H), 7.61 (t, J = 9.3, 2H), 6.91 (d, J = 8.7, 1H), 6.32 (d, J = 9.6, 1H), 4.26 (t, J = 6.9, 2H), 2.75 (m, 2H), 2.04 (t, J = 2.7, 1H). FTIR (cm⁻¹): 3417.24, 3243.68, 2358.51, 1710.55, 1681.62, 1598.70, 1560.13, 1486.85, 1394.28, 1295.93, 1241.93, 1182.15, 1118.51, 1079.94, 831.17, 767.53. ESI-MS: m/z = 265 [M + H⁺]. Melting point: 166–168 °C.

2.3. Preparation of 7-(but-3-yn-1-yloxy)-8-(difluoromethyl)-2H-chromen-2-one (AYF)

7-(But-3-yn-1-yloxy)-2-oxo-2H-chromene-8-carbaldehyde (2, 0.3671 g, 1.53 mmol) was dissolved in 7.65 mL anhydrous DCM, and subsequently diethylaminosulfur trifluoride (DAST) (0.56 mL, 4.58 mmol) was added and stirred for 24 h under argon atmosphere at room temperature. Afterwards, the reaction mixture was extracted with DCM/water. The organic layer was dried over MgSO₄. The solvent was removed and the residue was purified by flash chromatography on silica gel (ethyl acetate/n-hexane = 1 : 3) to obtain the title compound AYF as a light yellow solid (62%, 0.2496 g). ¹H NMR (CDCl₃, 300 MHz, ppm): δ = 7.62 (d, J = 9.3, 1H), 7.51 (d, J = 8.7, 1H), 7.26 (t, J = 53.1, 1H), 6.88 (d, 8.7, 1H), 6.30 (d, J = 9.6, 1H), 4.23 (t, J = 7.2, 2H), 2.74 (m, J = 11.4, 2H), 2.05 (t, J = 2.4, 1H). FTIR (cm⁻¹): 3303.46, 3075.9, 2958.52, 2900.41, 1735.62, 1608.34, 1498.49, 1390.42, 1319.07, 1249.65, 1191.79, 1118.51, 1074.16, 1006.66, 838.88, 779.10, 725.10, 636.39, 566.97, 466.69. ESI-MS: m/z = 264 [M + H⁺]. Melting point: 159–161 °C.

2.4. Assay conditions for the detection of Hg²⁺ using AYF

A stock solution of AYF was prepared in dimethyl sulfoxide (DMSO). In a typical assay, AYF (100 μM) and Hg²⁺ were incubated in DMSO/CBTP (pH = 7.6, 10 mM)/acetonitrile (ACN) (7 : 2 : 1, v/v/v) solution at the respective temperature and time. Various concentrations of standard Hg²⁺ ions or other metal ions (in the case of selectivity studies) were added, and the release of 7-hydroxy-8-formylcoumarin (coumarin 1) was monitored by recording fluorescence spectra at λ_ex = 360 nm and λ_em = 460 nm. The assay consists of two steps: (1) incubation of AYF in the solution containing DMSO and CBTP for 3 h at 90 °C and (2) ACN was added and incubated in a water bath for 1 h at 60 °C.

2.5. Assay conditions for the detection of Hg²⁺ using two-probe coupling assay (AYF and DPF₁)

The probe DPF₁ (7-((4-(tert-butyldiphenylsilyloxy)benzyl)-oxy)-8-(difluoromethyl)coumarin) was synthesized according to our previous report.²⁵ A stock solution of DPF₁ was prepared in ACN. The assay consists of two steps: (1) incubation of AYF (200 μM) and Hg²⁺ in 100 μL of DMSO/CBTP (pH = 7.1, 10 mM) (9 : 1, v/v) at 90 °C for 3 h and (2) addition of DPF₁ (50 μM) in ACN/pyridine (89 : 1, v/v) until the total volume of sample was 1 mL and incubation in a water bath for 3 h at 40 °C. Different concentrations of Hg²⁺ were added and the release of coumarin 1 was monitored.

2.6. Assay conditions for the detection of Hg²⁺ using AYF and DCC

A stock solution of DCC was prepared in ACN. The assay consists of two steps: (1) AYF (100 μM) and Hg²⁺ were incubated in the solution containing DMSO and CBTP (pH = 7.6, 10 mM) for 3 h at 90 °C and (2) DCC (5 μM) in ACN was added and incubated in a water bath at 60 °C for 1 h. The release of coumarin 1 was monitored at λ_ex = 360 nm and λ_em = 460 nm, while the release of 3-(2′-benzothiazolyl)-4-carbonitrile-7-hydroxycoumarin (coumarin 2) was monitored at λ_ex = 500 nm and λ_em = 600 nm.

2.7. Assay conditions for the detection of Hg²⁺ using three-probe coupling assay (AYF, DPF₁ and DCC)

The probe DCC (3-(benzothiazol-2-yl)-4-carbonitrile-7-((4-(tert-butyldiphenylsilyloxy)benzyl)oxy)coumarin) was prepared in accordance with our reported procedure.²⁵ Three-probe assay testing consists of two steps: (1) AYF (200 μM) and Hg²⁺ were incubated in 100 μL solution of DMSO/CBTP (pH = 7.1, 10 mM) (9 : 1, v/v) for 3 h at 90 °C and (2) DPF₁ (50 μM) and DCC (20 μM) in ACN/pyridine (89 : 1, v/v) were added and the whole solution (1 mL) was incubated in a water bath for 3 h at 40 °C. Various concentrations of standard Hg²⁺ ions were tested and the release of coumarin 2 was monitored.

3. Results and discussion

The synthesis of AYF is outlined in Scheme 3. A two-step synthesis from coumarin 1 gave the desired AYF. Coupling coumarin 1 with but-3-yn-1-yl-4-methylbenzenesulfonate under basic condition gave the alkylated ether 2 in 78% yield. Treatment of aldehyde 2 with DAST furnished the geminal di-fluorides and the resulting compound was named as AYF. The overall yield of the two steps was 48%. The chemical structures of the synthetic intermediates and the final products were determined by NMR and IR spectroscopy and mass spectrometry.

Scheme 3 Synthesis of AYF.

3.1. Detection of mercury with AYF

Under the same experimental conditions, the pristine analogue coumarin 1 is able to produce a highly intensive fluorescence signal (inset Fig. 1A), while AYF is unable to produce any signal. As expected, the fluorophore (coumarin 1) of AYF is completely masked by the specially designed trigger group. In the presence of Hg²⁺, AYF produced an obvious fluorescence signal corresponding to the emission spectrum of coumarin 1 (Fig. 1A). As explained in an earlier section, AYF undergoes oxymercuration in the presence of Hg²⁺ which unmasks and releases coumarin 1 which produces a fluorescence signal (Scheme 2). Furthermore, the sensing ability of AYF in the presence of different concentrations of Hg²⁺ was determined by recording its fluorescence spectra (λ_ex = 360 nm and λ_em = 460 nm) by following the assay conditions given in Section 2.4. The fluorescence intensity increases linearly as the concentration of Hg²⁺ increases. A plot between fluorescence intensity and concentration of Hg²⁺ exhibits a linear relationship in a concentration range of 2 ppm–10 ppm (Fig. 1B). The limit of detection (LOD) of AYF to detect Hg²⁺ was calculated to be 1.2 ppm. The LOD was calculated using the formula LOD = 3s_b/S, where s_b is the standard deviation of ten blank measurements and S is the sensitivity. The selectivity of AYF towards detection of Hg²⁺ in the presence of other metal ions was examined (Fig. 1C). AYF delivered well-defined fluorescence signal for 10 ppm Hg²⁺, whereas no significant fluorescence responses were observed for the other metal ions (Pd²⁺, Cu²⁺, Mg²⁺, Sr²⁺, Pb²⁺, Cd²⁺, Co²⁺ and Ni²⁺). Therefore, the probe AYF is suitable for Hg²⁺ detection in the presence of other metal ions.

Fig. 1 (A) Fluorescence enhancement (intensity difference I − I₀/a.u.) of AYF (100 μM) with Hg²⁺ (a = 0, b = 2, c = 4, d = 6, e = 8, f = 10 ppm) in 20% CBTP buffer (pH = 7.6, 10 mM) containing 70% DMSO and 10% can (inset: fluorescence emission spectra of AYF and pristine coumarin 1). I = intensity of the fluorescence signal at a particular concentration of Hg²⁺, I₀ = intensity of blank sample. (B) Linear calibration plot: I − I₀/a.u. vs. [Hg²⁺]/ppm. (C) Fluorescence emission change of AYF (100 μM) in the presence of other metal ions. The white bars represent the emission intensity in the presence of 10 ppm of the metal ion of interest. The black bars represent the change of the emission intensity in the presence of 10 ppm Hg²⁺ and 10 ppm of the metal ion of interest. Excitation was at 360 nm and the emission intensities were recorded at 460 nm. Error bars represent the standard deviation of three individual measurements.

3.2. Detection of mercury with AYF coupled with autocatalytic signal amplification (AYF + DPF₁)

In order to improve the sensitivity of AYF, we have incorporated a signal amplification strategy by using a two-probe coupling assay: AYF and DPF₁. The sensing ability of this coupling assay was determined by recording fluorescence spectra (λ_ex = 360 nm, λ_em = 445 nm) at different concentrations of Hg²⁺. The assay conditions were followed as given in Section 2.5. As shown in Fig. 2, the fluorescence intensity increases linearly as the concentration of Hg²⁺ increases and the plot between fluorescence intensity and [Hg²⁺] exhibits a linear relationship in a concentration range of 1–5 ppb. The LOD for Hg²⁺ using AYF coupled with DPF₁ is calculated to be 0.51 ppb which is more than 2000 times lower than that obtained for AYF alone. This must be due to the signal amplification through the cascade of self-immolative reactions which were induced by the latent generated fluorides as explained in Scheme 2. Thus, the fluorescence determination studies clearly revealed that the designed probes followed the proposed mechanistic pathway which rendered a low LOD as expected. It is worth mentioning that a previously reported fluorescein probe which was designed based on the reactivity of Hg²⁺ with alkynes was able to reach a low detection range of 8 ppb.²⁸ Interestingly, our two-probe coupling assay (AYF and DPF₁) incorporating a signal amplification strategy is able to reach a LOD of 0.51 ppb which is roughly 14 times lower than that of the previously reported fluorescein-based probe for Hg²⁺.

Fig. 2 (A) Fluorescence enhancement (intensity difference/a.u.) of two-probe coupling assay (AYF and DPF₁) with different concentrations of Hg²⁺ (a = 0, b = 1, c = 2, d = 3, e = 4, f = 5 ppb). (B) Linear calibration plot: I − I₀/a.u. vs. [Hg²⁺]/ppb. Error bars represent the standard deviation of three individual measurements.

In order to determine whether Hg²⁺ ions cleave DPF₁, we have carried out negative control experiments with DPF₁ alone in the presence and absence of Hg²⁺ (Fig. S7†). As seen from the figure, DPF₁ alone is unable to produce a significantly different fluorescence signal even in the presence of Hg²⁺ indicating that Hg²⁺ had no reaction with DPF₁. Hg²⁺ ions selectively cleave AYF and produce two fluoride ions which react with the DPF₁ and produce signal amplification. Therefore, the experimental results reveal that the designed probes follow the controlled sensing pathway as described in the schematic mechanism.

3.3. Mercury detection with long-wavelength fluorogenic probe

The use of a long-wavelength fluorogenic probe is an attractive alternative to conventional UV-visible fluorophores because fluorescence occurs in the long-wavelength region of the electromagnetic spectrum and can avoid auto-fluorescence³⁰ and minimize absorption or emission associated with other compounds that might be present in the sample. DCC is a fluorescent probe with maximum emission in the 595 nm region.³¹ Luminescence measurements in this region increase spectral selectivity because the interference of potential auto-fluorescence signals can be reduced.³² Using two small-molecule systems, AYF and DCC, we have examined the detection of Hg²⁺ in the long-wavelength region. The two-probe coupling assay (AYF and DCC) exhibits highly enhanced fluorescence signal for each addition of Hg²⁺ (Fig. 3A). The assay conditions for the detection of Hg²⁺ using AYF and DCC coupling assay are given in Section 2.6. The fluorescence intensity linearly increases as the concentration of Hg²⁺ increases in a range from 1 to 5 ppm (Fig. 3B). As explained in Scheme 2, the two geminal fluorides ejected by AYF induce self-immolative reactions of DCC to release long-wavelength reporter coumarin 2. Along with the advantage of long-wavelength detection, this assay platform has also shown higher sensitivity than the assay platform using only AYF. Therefore, the AYF and DCC coupling assay will be a promising approach for the selective and sensitive detection of Hg²⁺ in biological samples. In order to determine whether Hg²⁺ ions cleave DCC, negative control experiments with DCC alone in the presence and absence of Hg²⁺ were carried out (Fig. S8†). As expected, DCC alone in the presence of Hg²⁺ is unable to elicit a fluorescence signal indicating that Hg²⁺ had no reaction with DCC (Fig. S8†). Thus, DCC follows the controlled sensing pathway as described in the schematic mechanism.

Fig. 3 (A) Fluorescence emission changes (λ_ex = 360 and 500 nm), consisting of AYF (100 μM) upon incubation with different concentrations of Hg²⁺ (a = 0, b = 6, c = 8, d = 10 ppm) in 20% CBTP buffer (pH = 7.6, 10 mM) and 70% DMSO for 3 h at 90 °C, and 10% ACN solution of DCC (5 μM) was added for 1 h at 60 °C. (B) Linear calibration plot for two-probe coupling assay (AYF + DCC). Error bars represent the standard deviation of three individual measurements.

After successfully developing the long-wavelength assay with AYF and DCC, then we investigated the use of DCC probe coupled with AYF as a mercury-sensitive probe and DPF₁ as a signal amplification generator. The sensing ability of this three-probe coupling assay (AYF, DPF₁ and DCC) was determined by recording fluorescence spectra (λ_ex = 500 nm, λ_em = 595 nm) at different concentrations of Hg²⁺. The assay conditions were followed as given in Section 2.7. The intensity of fluorescence increases linearly as the concentration of Hg²⁺ increases and the plot between fluorescence intensity and [Hg²⁺] exhibits a linear relationship in a concentration range of 0.5–2.5 ppb (Fig. 4). The LOD for the three-probe assay is calculated to be 0.16 ppb (0.8 nM) which is lower than that for the two-probe assay (AYF and DPF₁) and around 7000 times lower than that obtained for AYF alone. Also, the LOD of our three-probe coupled assay is well below the allowed level of Hg²⁺ in drinking water regulated by the US EPA. Previously reported fluorescent probes based on rhodamine–coumarin conjugate, rhodamine dye attached with a receptor composed of thiol and alkene and Nile blue displayed LODs of 40 nM, 27.5 nM and 1 ppb, respectively.^33–35 In comparison with these previous probes, our three-probe coupling assay shows better sensitivity. Thus, the proof-of-concept of our bioinspired approach which uses Hg²⁺ as analyte and fluoride as signal transduction messenger as described in Schemes 1 and 2 is successfully validated. Although the sensor performance of our approach is not outstanding and requires the use of mixed solvents, it is interesting to develop novel approaches for Hg²⁺ detection by mimicking biological signal transduction pathways.

Fig. 4 (A) Fluorescence enhancement (intensity difference/a.u.) of three-probe coupling assay (AYF, DPF₁, and DCC) with different concentrations of Hg²⁺ (a = 0, b = 0.5, c = 1.0, d = 1.5, e = 2.0, f = 2.5 ppb). (B) Linear calibration plot: I − I₀/a.u. vs. [Hg²⁺]/ppb. Error bars represent the standard deviation of three individual measurements.

3.4. The selectivity of the developed platform

Our previous study has shown that the DPF₁ platform itself is highly specific for fluoride over other several common anions, including Cl⁻, Br⁻, I⁻, N₃⁻, CH₃COO⁻, HSO₄⁻, NO₃⁻, and H₂PO₄⁻.²⁵ Here, we have investigated the selectivity of our developed platform in the presence of other metal ions. Several other metal ions, including Pd²⁺, Cu²⁺, Mg²⁺, Sr²⁺, Pb²⁺, Co²⁺, Cd²⁺, Ca²⁺, Ba²⁺, Fe²⁺, Mn²⁺, Ni²⁺, Zn²⁺, Ag⁺, and K⁺, were tested (10 equivalents of Hg²⁺) (Fig. 5). No significant spectral changes were observed in the presence of other metal ions in comparison with blank sample, and only Hg²⁺ elicited a strong fluorescence signal. These results reveal the specificity of the detection platform for Hg²⁺. Moreover, our results are similar to those for another fluorescence-masking alkyl probe to detect Hg²⁺ which indicate that the presence of other metal ions will not significantly increase fluorescence intensity.²⁸

Fig. 5 Fluorescence emission change of three-probe coupling assay platform, consisting of AYF (200 μM), DPF₁ (50 μM), and DCC (20 μM), in the presence of other metal ions. The white bars represent the emission intensity in the presence of 1 ppm of the metal ion of interest (except for Mn²⁺, Ni²⁺ and Zn²⁺, 50 ppb). The black bars represent the change of emission intensity in the presence of 5 ppb Hg²⁺ and 1 ppm of the metal ion of interest (except for Mn²⁺, Ni²⁺ and Zn²⁺, 50 ppb). λ_ex = 500 nm and emission intensities were recorded at 595 nm. Error bars represent the standard deviation of three individual measurements.

3.5. Real sample analysis

We have explored the real-time applicability of our three-probe coupled approach for tap water and bovine serum samples via the spiking method. The spiked Hg²⁺ concentrations are 10 and 40 ppb in tap water sample, while they are 100 and 150 ppb in bovine serum sample. The detailed experimental procedure for preparation of tap water and bovine serum samples is given in the ESI.† The amount of Hg²⁺ in final experimental medium is calculated by considering the dilution made in the first step of the assay, wherein dilution factors of 20 and 200 were considered for tap water and bovine serum, respectively. The added, calculated, found and recovery values are given in Table 1. The quantitative recoveries of spiked samples were satisfactory, which reveals the promising practical applicability and validates the proof-of-concept of our approach inspired from biological signal transduction pathways.

Table 1 Determination of mercury present in tap water and bovine serum samples using three-probe platform (AYF, DPF₁, and DCC)

Sample	Spiked/ppb	Calculated amount of Hg^2+ in final experimental medium/ppb	Found/ppb	Recovery/%	RSD^a
a RSD of three individual measurements.
Tap water	10	0.5	10.81	108.09	0.77
	40	2	38.63	96.57	1.89
Bovine serum	100	0.5	94.73	94.73	1.89
	150	0.75	147.27	98.18	5.40

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4. Conclusions

In summary, we have successfully demonstrated the proof-of-concept of the AYF, DPF₁ and DCC coupled bioinspired fluorescence approach in the detection of Hg²⁺. The successful incorporation of the concept of a biological signal transduction strategy into the molecular design of probes was revealed by their fluorescence sensing ability towards Hg²⁺. The sensitivity of AYF to detect Hg²⁺ is greatly improved (about 7000 times) after it is coupled with DPF₁ and DCC which validates the significance of the described approach towards enhancing the sensitivity of Hg²⁺ detection. Besides, the real-time analyses demonstrated for water and biological samples add additional validation to the proof-of-concept. Despite the average sensing performance of the approach over benchmark Hg²⁺ fluorescence sensors, biomimic approaches have the advantage of nature and accomplish complex and multiple goals. The described bioinspired controlled multi-probe sensing approach holds great promise for the detection of metal ions and small molecules.

Acknowledgements

This work was supported by the Ministry of Science and Technology, Taiwan (NSC 103-2811-M-027-002, 102-2113-M-027-002-MY3, and MOST 104-2622-M-027-001-CC3).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11735j

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