Dye-Labeled Polyacryloyl Hydrazide-Ag Nanoparticle Fluorescent Probe for Ultrasensitive and Selective Detection of Au Ion.

The efficiency of a fluorescence sensing device based on metal-enhanced fluorescence (MEF) is dependent on the optimization of interaction between the fluorophore and the metal nanoparticle (NP). Herewith, ultrasensitive and selective turn-on sensing of Au3+ is achieved by using a suitable combination of fluorophore and metal NP system through sequential MEF effect. Dansyl hydrazide-tagged Ag NPs in the polyacryloyl hydrazide cavity are utilized to sense the picomolar concentration of Au3+ in aqueous media. We demonstrated that the selective Au3+ sensing is due to the selective deposition of Au on the Ag NP surface over the 16 other metal ions studied. The sensitivity is assigned to the strong overlapping of the emission band of the fluorophore with the surface plasmon band of the Au and improvement of fluorescence signal through successive MEF by Ag and Au colloids. The sensing is associated with a fivefold increase in fluorescence intensity and appearance of violet color of the solution. These luminescent Ag-Au bimetallic NPs may be utilized to trace cancer cells in biological systems and for cell imaging applications.

Introduction

Gold (Au) is precious to human being since ancient times because of its esthetic qualities. Au cations have also displayed promising medicinal qualities and are used in the treatment of tuberculosis and rheumatoid arthritis.[1] At the same time, the Au cations, especially Au3+, are reported to be toxic toward human[2] and aquatic species.[3] The above toxicity of soluble Au3+ is attributed to its selective binding ability toward DNA[4] and enzymes.[5] Au3+ is responsible for enzyme depletion and protein denaturation against selective cellular targets and lysosomal dysfunction, which subsequently results in DNA and membrane damage.[6] Au3+ also promotes the oxidative DNA damage by catalyzing the free-radical generation of various chemical entities frequently used in biochemical and biological studies.[7] The Au salts cause serious damage to kidney, liver, and peripheral nervous system.[8] Therefore, sensing tools to determine the concentration of Au3+ selectively with high sensitivity are desirable. In this regard, a number of reports on the sensing of Au cations in both aqueous[9] and organic[10] media are available in the literature. Sensing of Au ion in aqueous media is advantageous to detect the contamination in biological systems. Out of the various modes of sensing available for the detection of Au3+, the fluorescence mode is advantageous because of its operational simplicity and ultrasensitivity.[11] Au nanoparticles (NPs) are known to be strong fluorescence quenchers because of the associated energy-transfer processes;[12] therefore, turn-off fluorescence sensors for the detection of Au are reported in the literature. Turn-on sensors for Au3+ are also reported in the literature in which the Au3+ ion acts as a catalyst and chemically transforms the nonfluorescent probe to a fluorescent one.[13] However, the sensitivity is limited to micromolar concentrations because [Au3+] in above range is necessary to catalyze the sensing reaction.

NP-based probes for sensing applications have gained immense attention recently because of their high detection threshold,[14] low cost, fast response, high surface area, and portability.[15] A range of chemical,[16] optical,[17] electrochemical, biological,[18] and pH[19] sensors based on NPs are available in the current literature.[20] Nanosensors are promising especially in the area of biosensing because of the possibility of real-time and nonevasive monitoring of intracellular activities,[21] tracing disease biomarkers,[22] and toxic chemicals.[23] Metallic NPs possessing size- and shape-dependent optical properties or surface plasmon resonance are useful in colorimetric and fluorescence sensing applications.[24] Furthermore, because surface functionalization of these metallic NPs with different ligands alters their optical properties,[25] a variety of available surface-modified NPs broaden their applicability in the area of sensors. Dye labeling of the NPs is an attractive technique to impart unique emissive properties through plasmon-controlled fluorescence or metal-enhanced fluorescence (MEF) mechanism. MEF is dependent on the interaction between the fluorophore and the metal NPs, shapes and sizes of the metal NPs, and the distance between these NPs and the fluorophore. This enhancement is attributed to the radiative rate modification of the fluorophore in the close proximity of a metal NP.[26] As per the common understanding, strong overlapping of the excitation/emission band of the fluorophore with the surface plasmon band of the metal NP is anticipated to induce maximum fluorescence enhancement in the system.[27]

Therefore, careful selection of dye and metal NP system is important to design a highly sensitive fluorescence sensing device. Herewith, a dye-labeled Ag NP system stabilized by the polyacryloyl hydrazide (PAHz) cavity is designed to sense Au3+. PAHz is chosen as the stabilizing agent for multiple reasons. First, instant formation of Ag and Au NPs is possible in the PAHz solution under room-temperature conditions.[28] Second, covalent dye labeling of the PAHz-Ag NPs is possible by utilizing the swift reactivity of carbonyl hydrazide with a range of functional groups.[29] Last, the PAHz cavity around the metal NPs may act as a spacer layer and provide the much needed gap between the fluorophore and the NPs. Au is known to quench the fluorescence at shorter distances (<2 nm)[30] and exhibit MEF at longer distances (≥20 nm).[31,32] The procedure allows cost-effective and reagent-free one-pot synthesis of dye-labeled Ag NPs. Dansyl hydrazide (DH) possessing emission maximum (∼510 nm) similar to the surface plasmon band of Au NP (525 nm) and low (0.01) quantum yield (QY) is chosen as the fluorophore to offer strong interaction and achieve maximum sensing sensitivity through optimized MEF. Ag NP is anticipated to induce primary MEF to the DH in PAHz-DH-Ag NP. In the presence of Au3+, possible deposition of Au on Ag NP to form the corresponding Ag–Au bimetallic NP in the PAHz cavity is expected to improve the signal through a secondary MEF and to create the possibility of turn-on sensing of Au ion. Therefore, PAHz-DH-Ag NPs are synthesized, and their efficiency in sensing Au3+ at different concentrations in aqueous media is studied. The use of these highly fluorescence-enhanced bimetallic nano-objects in cell imaging and sensing of intracellular pH is accessed.

Experimental Section

Materials

Methyl acrylate (SD Fine Chemicals, >99%), potassium bromate (Merck, >99.0%), sodium hydrogen sulfite (Merck, 58.5–67.4%), acrylic acid (AA, Merck, >99.0%), sodium chloride (NaCl, Qualigens, >99.9%), hydrazine hydrate (SD Fine Chemicals, 99%), tetra-n-butylammonium bromide (TBAB, Merck, ≥98.0), silver nitrate (AgNO3, Qualigens, 99%), dansyl chloride (DC, Alfa Aesar, 97%), dimethyl sulfoxide (DMSO, SRL-chemicals), HAuCl4 (Sigma-Aldrich, 99.99%), CuSO4·5H2O (Fisher Scientific, 98.5%), Co(NO3)2 (Qualigens, >99.9%), CdCl2 (Qualigens, >95%), HgCl2 (Fisher Scientific, 99.9%), SnCl4 (Fisher Scientific, 99.9%), FeSO4·7H2O (Fisher Scientific, 99.9%), TiO2 (Qualigens, >99.9%), NiCl2 (Qualigens, >99.9%), Cr2(SO4)3 (Qualigens, >99.9%), MgSO4 (Merck, 99%), PbCl2 (Qualigens, >99.9%), ZnSO4·7H2O (Qualigens, >99.0%), platinum bromide (PtBr2, Sigma-Aldrich, 98%), and cesium chloride (CsCl3·7H2O, Qualigens, >99.9%) were used without further purification. Tetrahydrofuran (THF, Qualigens, 99.0%) was refluxed over sodium metal and benzophenone overnight and distilled under a nitrogen atmosphere prior to use. Water used for the preparation of aqueous solution was obtained by deionization and filtration with a Millipore Elix-10 purification apparatus. The electrical conductivity of deionized water is 0.0054 mS/cm.

Characterization

A LabIndia UV–VIS 3200 instrument was used to record the ultraviolet–visible (UV–vis) spectra of the sample. UV–vis spectra were recorded at 1 nm/min scan rate. A Cary Eclipse fluorescence spectrophotometer (serial no. MY14270004) was used to record the fluorescence spectra of the sample. The spectrophotometer uses a xenon flash lamp for superior sensitivity, high signal-to-noise ratio, and fast kinetics. Fluorescence spectra were recorded at every 12.5 ms and scanned at 24 000 nm/min without peak shifts.

Field-emission scanning electron microscopy (FESEM) images were recorded using a Carl Zeiss-Sigma field-emission microscope operating at an acceleration voltage of 3 kV. Samples were prepared by drop-casting the polymer solution on a silver foil. The samples were sputtered with gold before recording the images.

Transmission electron microscopy (TEM), high-resolution TEM (HRTEM), and high-angle annular dark-field scanning TEM (STEM) data were recorded on the JEOL JEM-2100 and FEI Tecnai G2 F20 electron microscopes operating at 200 kV. For the TEM and HRTEM measurements, a drop of the PAHz-DH-Ag NP and PAHz-DH-Ag–Au NP solution was dispensed onto a 3 mm carbon-coated copper grid. Excess solution was removed with an absorbent paper, and the sample was dried under vacuum at room temperature. An energy-dispersive X-ray spectroscopy (EDX/EDS) analyzer attached to the TEM operating in the STEM mode was used to analyze PAHz-DH-Ag NPs and PAHz-DH-Ag–Au NPs. The selected-area electron diffraction (SAED) and ring diffraction patterns of PAHz-DH-Ag–Au NPs were analyzed using a CrysTBox tool.[33]

The size distribution curves of the particles present in the solutions were measured by the dynamic light scattering (DLS) approach using a Zetasizer Nano-ZS instrument (Malvern) equipped with a green laser (523 nm). The intensity of scattered light was detected at an angle of 173.1°. For each sample, three measurements were performed. The data processing was carried out using the Zetasizer software 7.10 (Malvern Instruments). The size distributions were reported as volume and number distributions.

Time-resolved fluorescence intensity decays were obtained using a commercial time-correlated single photon counting setup (LifeSpec II, Edinburgh Instruments, U.K.). All samples were excited at 376 nm, and the full width at half-maxima of the instrument response function (IRF) is 110 ps. For lifetime measurements, peak counts of 4096 were collected with the emission polarizer oriented at magic-angle polarization, and decays were collected at 506 nm. The time-resolved fluorescence intensity decays were analyzed by deconvoluting the observed decays with the IRF to obtain the intensity decay function, manifested as a sum of three exponentials for the present study.[34]

Synthesis of PAHz

PAHz was synthesized following a previously reported procedure.[28]

Synthesis of PAHz-DH

To an aqueous solution of PAHz (0.02 g/mL) in 2 mL of deionized water, DC (0.47 mM) was added at room temperature, and the mixture was stirred for 40 min. The progress of the reaction was monitored by UV–vis and fluorescence spectrophotometer. The plateauing of fluorescence intensity at 506 nm was considered to be completion of the reaction and formation of PAHz-DH.

Synthesis of PAHz-DH-Ag NPs

A typical synthetic procedure is described below. The aqueous solution of PAHz-DH (0.02 g/mL) and AgNO3 (30 μM) was mixed together and stirred at room temperature for 30 min. The color of the solution turned to light yellow, suggesting the PAHz-DH-Ag NP formation. The characterizations of PAHz-DH-Ag NPs were done by using UV–vis, fluorescence, TEM, DLS, and SEM analyses.

Sensing of Different Metal Ions

To confirm the selectivity of the sensor, various metal ions (Au3+, Cs3+, Ca2+, Co2+, Cu2+, Cd2+, Fe2+, Mg2+, Ni2+, Zn2+, Ag+, Pt2+, Hg2+, Sn2+, Cr3+, Ti4+, and Pb2+) (20–800 pM) were added to an aqueous solution of PAHz-DH-Ag NP (0.02 g/mL), and the fluorescence was recorded after 40 s of the addition of metal ions. The change in intensity versus metal ion concentration was monitored to understand the sensing efficiency of the PAHz-DH-Ag NPs.

Culture and Maintenance of HEK293 Cells

HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cells were supplemented with an antibiotic–antimycotic solution (100 units penicillin, 0.1 mg mL–1 streptomycin, and 0.25 mg mL–1 amphotericin B) and grown at 37 °C under standard cell culture conditions (5% CO2, 95% humidity). HEK293T cells were adhered to the cover slip for 24 h before the study.

Fluorescent Confocal Imaging

HEK293 cells after 20 min incubation with dye-labeled Ag or Ag–Au NPs at pH 8.0 or 5.4 with appropriate controls were washed with 1× phosphate-buffered saline (PBS) and were fixed with 4% paraformaldehyde for 20 min at 40 °C. Imaging was done using an Olympus BX61-FV1200-MPE microscope equipped with a 405 nm laser using 40× oil (1.3NA) objective with 8.0 μs/pixel scan speed or by a fluorescent microscope Leica DMI6000 20× objective. Cropped images were assembled for presentation in Adobe Photoshop (version 7.1).

XTT Assay

HEK293 cells were grown in complete culture media (DMEM with 10% FBS) in 5% CO2 and 95% humidity at 37 °C. The cells were seeded into 96-well plates at a density of 50 000 cells per well in culture media, and then 1 μM, 500 μM, and 1 mM concentrations were added in quadruplicates of PAHz-DH-Ag–Au NPs. The cells were incubated with the NPs for 20 min after which the medium was removed and fresh medium was added along with the kit reagents as followed in the kit protocol (cell proliferation kit II, Roche, Cat no. 11465015001). After 6 h incubation with the reagent, absorbance was measured using an ELISA plate reader at 480 nm.

Fluorescent Imaging

HEK293 cells after 60 min preincubation with Au NPs with appropriate controls were washed with 1× PBS and were fixed with 4% paraformaldehyde for 20 min at 40 °C. Imaging was done using a fluorescent microscope Leica DMI6000 20× and 40× objective. The dye was excited using filters with excitation in the range of 340–380 nm, and emission was collected at 425 nm. The Au NP preincubated HEK293 cells were further incubated for 30 min with PAHz-DH-Ag NPs. The cells were washed with 1× PBS and were fixed with 4% paraformaldehyde for 20 min at 40 °C, and images were recorded.

Results and Discussion

DH was covalently labeled to PAHz by reacting DC (CONHNH2/DC = 500:1, mol/mol) with PAHz in aqueous solution at 25 °C (Figure ). Appearance of an absorption maximum (λmax) at 330 nm after 5 min of reaction time suggested the formation of PAHz-DH (Figure A). The intensity of emission maxima at 506 nm gradually increased with time and reached a plateau after 30 min, suggesting the completion of reaction (Figure B).a

Figure 1

Schematic representation of DH labeling to PAHz followed by the formation of Ag and Ag–Au NPs and the MEF of DH by the NPs.

Figure 2

(A) UV–vis and (B) fluorescence traces of PAHz (0.02 g/mL) and DC (0.23 mM) in aqueous solution recorded after regular time intervals showing the gradual formation of PAHz-DH. Inset B: Variation of intensity at 506 nm with time. The effect of [Ag+] and [PAHz] on the (C) absorption maxima and (D) fluorescence intensity of DH in the resulting PAHz-DH-Ag NPs. [PAHz] = 0.02 g/mL and [DC] = 0.48 mM were maintained in the preparation of all the compositions. All the spectra were recorded after 30 min of the addition of Ag+ to PAHz-DH solution. The excitation wavelength was 325 nm for recording the fluorescence spectra. The fluorescence was monitored in the 335–650 nm range. The absorbance and intensity values presented in “C” and “D”, respectively, are the average of five experiments and are provided with error bars.

The PAHz-DH-Ag NPs were synthesized by the in situ reduction of Ag+ in the PAHz-DH solution by the CONHNH2 functionality and capping of the resulting Ag NP with CONHNH2 as similarly reported recently.[28] The λmax at 410 nm attributable to the surface plasmon band of Ag NP appeared within 5 min of the addition of Ag+ at 25 °C and reached a plateau by 30 min of reaction time (Supporting Information, Figure S1). The fluorescence intensity at 506 nm simultaneously increased with the formation of Ag NP, suggesting fluorescence enhancement of DH by the Ag NP. Because the MEF effect is sensitive to the thickness of the spacer layer and the concentration of the metal NPs, the enhancement factor was optimized by varying the [Ag+] and [PAHz] in solution. For a fixed [PAHz], the absorbance at 410 nm gradually increased with [Ag+], suggesting an increase in Ag NP amount in solution (Figure C, Supporting Information, Figure S2). The fluorescence intensity was maximum for the PAHz-DH-Ag NPs prepared using 0.02 g/mL PAHz in solution (Figure D, Supporting Information, Figure S3). For any particular [PAHz] in between 0.01 and 0.1 g/mL, optimum intensity was noticed in the presence of 15–50 μM of [Ag+]. The fluorescence intensity of the samples strongly decreased on increasing the [Ag+] beyond 50 μM. Therefore, the PAHz-DH-Ag NPs synthesized using 0.02 g/mL [PAHz], 0.48 mM DC, and 30 μM [Ag+] were used for further studies.

The above NPs were mostly spherical in nature with diameters in the range of 10–20 nm (Figure A). The average particle size (davg) of the PAHz-DH-Ag NPs in solution was ∼61 nm with a polydispersity index value of 0.3 (Figure B, Supporting Information, Figure S4). The PAHz cavity thickness around the Ag NP was approximated to be half the difference between davg values obtained from the DLS and TEM analyses and found to be ∼25 nm. The FESEM analysis of the PAHz-DH-Ag NPs displayed uniform particles with diameters in the range of 40–80 nm, supporting the DLS data (Supporting Information, Figure S5).

Figure 3

(A) TEM image of PAHz-DH-Ag NP synthesized using 0.02 g/mL [PAHz], 0.48 mM DC, and 30 μM [Ag+]. Inset A: Particle size distribution of NPs. (B) DLS traces of PAHz-DH-Ag NP solution in the presence of various metal ions (300 pM).

Various metal salts in picomolar amounts were added to the aqueous solution of PAHz-DH-Ag NP, and the change in spectroscopic properties was monitored. In the presence of 40 pM Au3+ in the PAHz-DH-Ag NP solution, a λmax at 537 nm accountable to the surface plasmon band of Au NP appeared, suggesting the formation of Ag–Au bimetallic NP (Figure A). The increase in absorbance of the λmax at 537 nm with Au3+ amount and the isosbestic point at 465 nm supported the gradual deposition of Au on the Ag NP surface. The TEM image displayed the d-spacing values for both Ag (2.22 Å) and Au (2.35 Å) and supported the formation of Ag–Au bimetallic NP (Figure B). The spherical shape of the Ag NPs changed to prism on the formation of Ag–Au bimetallic NP. The UV–vis trace of the resulting PAHz-DH-Ag–Au NP was similar to that of the Ag–Au nanoprism reported earlier.[35] The EDS elemental mapping revealed the presence of both Ag and Au in the NP, and the overlay mapping suggested the presence of Au on the surface (Figure C–E, Supporting Information, Figure S6). The ratio between Ag and Au was measured to be ∼99.92:0.08 (w/w) (Supporting Information, Figure S7).

Figure 4

(A) UV–vis traces of PAHz-DH-Ag NP (0.02 g/mL) in the presence of different amounts of Au3+, (B) TEM image of PAHz-DH-Ag–Au NP, EDS elemental mapping of (D) Ag, (E) Au, and (C) overlay mapping of PAHz-DH-Ag–Au NPs prepared using 0.02 g/mL PAHz-DH-Ag NP and 300 pM Au3+.

The davg value of the PAHz-DH-Ag NP increased to 80 nm in the presence of 300 pM Au3+, supporting the formation of the above bimetallic NP (Figure B). The formation of the corresponding Ag–Au NP is associated with the oxidation of carbonyl hydrazide functionality of PAHz. However, because the amount of CONHNH2 functionality (mM) present in the solution is ∼1010 times higher than that of Au3+ (pM) added to the solution, the oxidative change may be negligible, and the PAHz cavity thickness is assumed to remain unaltered during the NP formation process. The davg gradually increased from ∼63 to ∼119 nm on increasing [Au3+] and from 25 to 800 pM in the PAHz-DH-Ag NP (0.02 g/mL) solution, suggesting that the amount of Au3+ in the solution controlled the Au thickness in the resulting Ag–Au bimetallic NPs (Supporting Information, Figure S4). The SAED image supported the presence of both the metals and the crystalline nature of the bimetallic NPs (Supporting Information, Figure S8).

To realize the possibility of the Ag–Au alloy NP formation using the procedure, both Ag+ and Au3+ in different molar proportions were added together to the PAHz solution (0.02 g/mL), and the mixture was incubated at 40 °C for 2 h. The UV–vis analysis revealed a single plasmon band for each of the compositions (Figure A). The λmax gradually red-shifted from 425 to 545 nm on increasing the Au3+ content in the reacting mixture, suggesting the formation of the Ag–Au alloy NP (Figure B). The dependence of absorption maxima on the ratio between Ag and Au content in alloy NPs is well-documented in the literature.[36,37] The absorbance values of the Ag–Au alloy NPs were lower than that of the Ag and Au NP, which supported the theoretical values calculated by using the Mie equation.[38]

Figure 5

(A) Surface plasmon band of the PAHz-DH-Ag–Au alloy NP synthesized by adding different molar proportions of Ag+ and Au3+ together (60 μM) to the PAHz-DH solution (0.02 g/mL). The spectra were recorded after incubating the mixture at 40 °C for 2 h. (B) Effect of Au3+ mol fraction in Ag+ and Au3+ mixture on the absorption maxima of the corresponding PAHz-DH-Ag–Au alloy NP.

The fluorescence signal of DH further improved with increase in [Au3+], suggesting successive MEF by the Ag–Au NP (Figure A). A noticeable increase in fluorescence intensity at 506 nm occurred in the presence of 40 pM Au3+ within ∼5 s. The intensity value reached a plateau within 40 s of the addition of Au3+ (Supporting Information, Figure S9). Recently, a boron-dipyrromethene (BODIPY) Schiff base has been reported for instant turn-on detection of Au3+ in the nanomolar concentration.[39] Nanoporous alumina-based interferometric sensor is also used for instant detection of micromolar amount of Au3+.[8] The current procedure allows real-time monitoring of Au3+ and higher sensitivity compared to that of the existing procedures. Optimum enhancement (∼fivefold) was noticed for [Au3+] in the range of 100–300 pM (Figure B). The color of the solution also appeared to be green under UV light, suggesting that these PAHz-DH-Ag NPs may be used for fluorescence sensing of picomolar amount of Au3+ in turn-on fashion (Figure C). A range of other metal ions in molar proportions (100 pM) similar to that of Au3+ were also added to the PAHz-DH-Ag NP solution, and fluorescence data were recorded after ∼40 s of incubation period. The fluorescence enhancement was selective to Au3+ only among the 17 metal ions studied (Figure D, Supporting Information, Figure S10). The interference of other metal ions toward the sensing of Au3+ was investigated by adding Au3+ (100 pM) to the PAHz-DH-Ag NP solution (0.02 g/mL) along with another metal ion (100 pM) in the same molar proportion. The UV–vis and fluorescence traces remained mostly unaffected by the presence of other ions, suggesting that the sensing is robust and insensitive toward the presence of other metal ions. In fact, the sensing of Au3+ was equally efficient in the presence of multiple metal ions (Supporting Information, Figure S11). The interference of other metal ions toward the davg value of the resulting PAHz-DH-Ag–Au NP was also negligible, suggesting that the deposition of Au on the Ag NP was least affected by the presence of other metal ions in solution (Supporting Information, Figure S12).

Figure 6

(A) Fluorescence spectra of PAHz-DH-Ag NP solution (0.02 g/mL) in the presence of different Au3+ concentrations. The excitation wavelength was 325 nm. The fluorescence was monitored in the 335–650 nm range. (B) Effect of [Au3+] on the fluorescence intensity of PAHz-DH-Ag NP (0.02 g/mL). The intensity values reported are an average of five experiments. (C) Color of the PAHz-DH-Ag NP solution under UV light in the presence of 75 pM metal ions and under visible light in the presence of 450 nM metal ions, (D) variation in fluorescence intensity at 506 nm of the PAHz-DH-Ag NP solution in the presence of different metal ions (100 pM). The incubation time was 40 s for all the metal ions. The values in “B” and “D” are provided with error bars.

The ultrasensitivity of PAHz-DH-Ag NPs toward Au3+ may have resulted from a strong interaction between the dye and the NP through the matching of surface plasmon band of the Au NP (537 nm) with that of the emission band of DH (506 nm).[40] Spectral overlap between the fluorophore and plasmon-resonant metal NPs is known to reinforce their fluorescence properties.[41] The color of the PAHz-DH-Ag NP solution (0.02 g/mL) turned green under UV light within 5 s of the addition of 75 pM or higher Au3+ (Figure C). The QY = Krad/(Krad + Knon-rad) of the resulting PAHz-DH-Ag–Au NP increased to 0.49 from 0.07 (PAHz-DH-Ag NP) (Figure , Supporting Information, Table S1). Interestingly, the average fluorescence lifetime [τ = 1/(Krad + Knon-rad)] of the PAHz-DH-Ag–Au NPs (∼13.1 ns) also experienced a sevenfold increase compared to that of the PAHz-DH-Ag NP (1.8 ns). Possibly, the replacement of Ag with Ag–Au NP has not substantially altered the radiative decay rate (Krad) of the fluorophore. Other quenching processes including aggregation of fluorophores may also be minimal because of the covalent attachment of the dye to the PAHz cavity and fairly low concentration of DH (DH/CONHNH2 = 1:500, mol/mol) in the solution. Therefore, the QY and τ changed proportionally to each other.

Figure 7

Fluorescence decay profile of PAHz-DH-Ag and PAHz-DH-Ag–Au NPs. Inset: QY and average lifetime (τ) data of both the NPs.

We have previously shown that PAHz-Ag NPs are pH-responsive and are capable of penetrating and delivering drugs in cancer cells.[28] The pH-responsive fluorescent properties of a porphyrin moiety in the vicinity of PAHz-Ag NPs have also been reported recently.[42] The pH responsiveness of the optical properties of the luminescent PAHz-DH-Ag–Au bimetallic NPs synthesized in this report was investigated to explore their utility in cell imaging and tumor detection applications. Because the pH of the cancerous cell environment is ∼5.4, the UV–vis and fluorescence spectra of the PAHz-DH-Ag–Au NP were recorded under pH 5.4 conditions and compared to that of the data at pH 8.0 (pH of 0.02 g/mL PAHz). The fluorescence intensity of the PAHz-DH-Ag–Au NP solution exhibited ∼12-fold decrease on changing the pH from 8.0 to 5.4, whereas the change in the absorbance value associated with the above change in pH was marginal (Figure A,B). The davg value decreased by ∼43% on changing the pH to 5.4, suggesting that the PAHz cavity thickness around the Ag–Au NP shrank under the acidic pH (Figure C). The above change in the spacer thickness may have affected the MEF of the fluorophore in the system (Figure B). To understand the intracellular behavior of the PAHz-coated Ag–Au NP, both PAHz-DH-Ag and PAHz-DH-Ag–Au NPs were incubated for 20 min in HEK293 cells under pH 8.0 and 5.4 conditions, and the confocal images were acquired. The fluorescence of PAHz-DH-Ag–Au NPs substantially increased compared to that of the PAHz-DH-Ag NPs in the cells, suggesting that the former may be used for cell imaging applications (Figure D1,D2). More importantly, the fluorescence of PAHz-DH-Ag–Au NPs notably decreased on changing the pH from 8.0 to 5.4 (Figure D3). These results suggested that PAHz-DH-Ag–Au NPs act as a pH-dependent fluorescent sensor, which may have potential for the identification of solid tumors. We also performed the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay to see the effect of NPs on cytotoxicity. We used PAHz-DH-Ag–Au NPs for this assay because these have the highest concentration of metals. No cytotoxicity on HEK293 cells was noticed for NPs loading up to 500 μM (Figure E). The HEK293 cells preincubated with Au3+ for 1 h were exposed to the PAHz-DH-Ag NP probe.

Figure 8

(A) UV–vis, (B) fluorescence spectra, and (C) DLS traces of PAHz-DH-Ag–Au NP solution under different pH conditions; the excitation wavelength for the fluorescence spectra was 325 nm. The fluorescence was monitored in the 335–650 nm range. Confocal images of the HEK293 cells after incubation with (D1) PAHz-DH-Ag, (D2) PAHz-DH-Ag–Au NPs at pH 8.0, and (D3) PAHz-DH-Ag–Au NPs at pH 5.4 conditions, and the images were acquired using 40× objectives and a 405 nm laser. (E) XTT assay of the PAHz-DH-Ag–Au NPs. The experiment was conducted in quadruplicates, and the data show the mean ± SEM of the absorbance at 480 nm. *p < 0.05 compared to vehicle by one-way ANOVA followed by Newman–Keuls post hoc analysis. Statistical analyses were performed using the Graphpad Prism 5 software.

As anticipated, the fluorescence of the probe increased in the cells, suggesting that these PAHz-DH-Ag NPs may be used for the intracellular detection of Au3+ (Supporting Information, Figure S13). These preliminary results suggest PAHz-DH-Ag–Au NP to be an important fluorescent biosensor for detecting the change in pH in cellular environments, which might have diagnostic potential for solid tumors.

Conclusions

The dye-labeled PAHz-capped Ag NP can be used as the reactive probe for instant fluorescent detection of picomolar amount of Au3+ in aqueous and biological environments. In the presence of Au3+, the PAHz-DH-Ag NP converts to the PAHz-DH-Ag–Au bimetallic NP, and the fluorescence signal enhancement of DH occurs owing to strong MEF by Au. The PAHz cavity thickness around the metal NP and the covalent attachment of the dye play important roles in optimizing the signal enhancement of the fluorophore. These bimetallic NPs are pH-responsive and promptly change their size in response to change in pH, which alters their optical properties. Owing to the above conditions, these fluorescent-enhanced PAHz-DH-Ag–Au NPs may be used for cell imaging as well as optical detection of solid tumors. These bimetallic NPs are cytocompatible up to 500 μM loading. This procedure may be extended to synthesize Ag–Au alloy NPs. In future, drug may be loaded in these luminescent pH-responsive alloy NPs for simultaneously diagnostic as well as therapeutic applications.