Fluorescence turn-off detection of spermine in biofluids using pepsin mediated synthesis of gold nanoclusters as a probe

Journal of Molecular Liquids 280 (2019) 18–24

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Fluorescence turn-off detection of spermine in biofluids using pepsin mediated synthesis of gold nanoclusters as a probe Jigna R. Bhamore a, Z.V.P. Murthy b, Suresh Kumar Kailasa a,⁎ a b

Department of Applied Chemistry, S. V. National Institute of Technology, Surat 395 007, India Chemical Engineering Department, S. V. National Institute of Technology, Surat 395 007, India

a r t i c l e

i n f o

Article history: Received 10 December 2018 Accepted 24 January 2019 Available online 28 January 2019 Keywords: Spermine P-Au NCs Fluorescence detection Spectroscopic and microscopic techniques

a b s t r a c t In this work, a simple, one step and eco-friendly synthetic strategy is developed for the fabrication of gold nanoclusters (Au NCs) using pepsin as a reducing and capping agent. The pepsin-Au NCs (P-Au NCs) displayed the excitation and emission peaks at 416 and 655 nm, and exhibiting red luminescence under UV lamp at 365 nm. The fluorescence quantum yield (QY) of P-Au NCs is 7.4%. The P-Au NCs act as specific biosensor for the detection of spermine in the presence of other structural similar compounds via fluorescence turn-off mechanism. The sensing principle is based on the spermine-induced fluorescence quenching of P-Au NCs, which leads to increase the size of Au NCs. The degree of florescence quenching was linear with spermine concentration (0.0075–10 μM), with a detection limit of 1.75 nM, which is much lower for the quantification of spermine in urine samples of cancer patients. Compared to other reported methods, the P-Au NCs-based analytical strategy provides a simple synthetic route for fluorescent Au NCs with good QY and showed high selectivity for the detection of spermine with improved accuracy and precision. This method demonstrates as a facile and cost-effective analytical strategy for sensitive and selective fluorescence turn-off assay of spermine in urine samples. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Spermine is a biogenic aliphatic tetramine (polyamine) that has widely found in all eukaryotic cells [1]. It exists as a polycation at physiological condition, and has structural similarities with spermidine, putresciene and cadaverine. Importantly, it acted as a potential biomarker for the diagnosis of cancer because its concentration variations in urine of normal and cancer patients, which indicates the presence of malignant tumors [2]. It has been involved in various biochemical pathways in cells such as gene regulation, inhibition of DNA damage, chromatin stabilization, and the preclusion of endonuclease-mediated DNA crumbling, respectively [3,4]. It has been noticed that the levels of spermine are very low in the blood samples of chronic kidney disease and strokes, whereas high levels of spermine are found in urine, saliva and liver of breast cancers patients [5]. Therefore, the quantification of spermine in biofluids (urine, saliva and blood) may be provided useful information for the rapid diagnosis of cancer disease. In view of this, the development of simple analytical strategy for the quantification of spermine in biofluids has received significant interest in screening of the tumors stages in various types of cancer diseases, which can significantly useful for monitoring of tumors growth and diagnosis of cancer. At present, few analytical tools such as spectrometry [6], ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (S.K. Kailasa).

https://doi.org/10.1016/j.molliq.2019.01.132 0167-7322/© 2019 Elsevier B.V. All rights reserved.

mass spectrometry [7], chromatographic [8,9], electrophoresis [10] have been used for the detection of polyamines including spermine. These techniques are comparatively time consuming and required expensive instrumentation with expert operator. Many reports have already described the use of UV–visible absorption and fluorescence spectrometric techniques for the detection of spermine by using various chemical composites such as chromophores [11,12], organic polymer [4], coordination compounds [13], and nanoparticles [14,15] as probes or signalling units. Moreover, these methods are essentially needed tedious purification steps and chemical derivatizations steps for producing the color or quenching/enhancing emission intensities in the presence of spermine. Recently, quantitative analysis of biologically important molecules by using fluorescent probes has become one of the promising approach in the field of analytical and bioanalytical sciences [16–19]. Among these, metal NCs have distinctive optical properties than the metal nanoparticles, which is due to their quantized energy states and molecular like electronic structure [20]. Fluorescent Au NCs have been recognized as stimulating probes in the field of sensing, imaging and drug delivery because of their unique properties i.e., ultra-small size, large Stokes shift, excellent photostability, and longer fluorescence lifetimes, respectively [21]. Notably, the free electron model was observed in the fluorescent Au NCs due to their intraband (sp–sp) transitions. The energy level spacing of intraband is strongly dependent on the size of the clusters [22]. Metal NCs have shown significant applications in bio- sensing and imaging because of their dispersibility, non-toxic behaviour and excellent

J.R. Bhamore et al. / Journal of Molecular Liquids 280 (2019) 18–24

biocompatibility [22]. Compared to silver and copper NCs [23–26], Au NCs have been recognized as popular and promising agents in various applications because of their easy preparation, whereas other metal NCs required specific reducing agents (hydrazine hydrate and sodium borohydride) for the reduction of Ag+ and Cu2+ ions. Until now, fluorescent Au NCs have been fabricated by using various biopolymers (proteins) as reducing agents via one-step reactions. For example, Xie et al., synthesized red luminescent Au NCs by using bovine serum albumin (BSA) as a ligand [27]. Significant research activities have been illustrated on the facile one-step synthesis and applications of Au NCs using various biopolymers i.e., lysozyme [28,29], horseradish peroxidise [30], lactotransferrin [31], human serum albumin [32,33], trypsin [34], pepsin [35], and egg white [36] as reducing agents. In this addition, our group synthesized red luminescent Au NCs using protein mixture (BSA and bromelain) to tune the fluorescence properties for sensing of Hg2+ ion and lambda cyhalothrin [37]. These studies reveal that the development of a facile synthetic approach for the fabrication of water-dispersible and stable Au NCs is of important significance but challenging. Herein, we report the green synthesis of P-Au NCs by using pepsin as a potential reductant and stabilizing agent. The as-fabricated P-Au NCs show maximum emission peak at 655 nm when excited at 416 nm. Noticeably, there is no surface plasmon resonance peak in the visible range (510–530 nm), signifying the size of P-Au NCs is b3.0 nm. The fabricated P-Au NCs have successfully demonstrated as a nanosensor for the fluorescence “turn-off” detection of spermine in biofluids. The emission intensity of P-Au NCs at 655 nm was drastically quenched by the addition of spermine. With increasing concentration of spermine, the fluorescence intensity ratio (I0/I) at 655 nm exhibited good linearity in the range of 0.0075–10 μM (R2 = 0.9904) with a detection limit of 1.75 nM. The use of pepsin as a ligand not only reduce Au3+ ion to Au0 NCs but also allows to develop a novel nanoprobe for the detection of spermine as a cancer biomarker in biofluids. Thus, P-Au NCs designed fluorescent probe may provide significant impact and serve as alternative analytical strategy for the specific detection of spermine in biofluids.

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(1:3, HCl:HNO3) and then rinsed with the distilled water and acetone prior to use. The luminescent P-Au NCs were synthesized by using pepsin as a ligand. In brief, 225 mg of pepsin was transferred into a reaction flask that contains 10.0 mL of Milli-Q water. To this, 0.5 mL of 100 mM HAuCl4 solution was added and stirred for 2.0 min. Then, 1.0 M of NaOH solution was added to the above solution to reach the pH 11.6 and the mixture solution was stirred continuously for 3 h at room temperature, which yields the color of solution from light yellow to light brown color. The formation of P-Au NCs was confirmed by absorption, fluorescence and FT-IR spectroscopic techniques. 2.4. Fluorescence turn-off detection of spermine Various concentrations of spermine (0.0075–100 μM) solutions were prepared by diluting 500 μM of spermine stock solution (5.1 mg/25 mL). For the fluorescence turn-off detection of spermine, 0.5 mL of spermine with different concentrations (0.0075–100 μM) was mixed with 1.0 mL of P-Au NCs solutions separately and the sample vials were vortexed for 20 min. The emission spectra of the above samples were measured at excitation wavelength of 416 nm. To evaluate the selectivity of the probe, different kinds of -NH2 group containing biomolecules (histidine, glutathione, cysteine, methionine, spermidine, adenine, isatin, and thymine, 500 μM) were added separately into P-Au NCs solutions and measured their emission spectra at excitation wavelength of 416 nm. 2.5. Detection of spermine in biological sample The biofluids (human plasma and urine) samples were obtained from Iyer laboratory, Surat, India and the detection of spermine in biofluids was followed by the literature [38]. Prior to detection, the collected biofluids were diluted to 100-fold using water and then spiked with different concentrations of spermine (10, 50 and 100 nM) and incubated for 30 min. The emission spectra of corresponding solutions were measured at excitation wavelength of 416 nm. The amount of spermine was quantified by the above described procedure.

2. Experimental section 3. Results and discussion 2.1. Chemicals and materials 3.1. Synthesis and characterization of P-Au NCs Hydrogen tetrachloroaurate hydrate (HAuCl4·xH2O), sodium hydroxide (NaOH), and metal salts (Mn(NO3)2·4H2O, FeCl3·6H2O, Pb(NO3)2, NiSO4·6H2O, Co(NO3)2·6H2O, Cu(NO3)2·3H2O, Cd(NO3)2· 4H2O, Mg(NO3)2·6H2O, FeCl2·4H2O and Zn(NO3)2·6H2O) were purchased from Sigma Aldrich, USA. Pepsin was obtained from Enzyme Bio Science PVT LTD, India. All biomolecules were purchased from Sigma Aldrich, USA. Water was prepared from a Milli-Q system and used for all experiments. 2.2. Instrumentation Fluorescence spectra were recorded on a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, USA), equipped with the Xenon flash lamp. UV–visible absorption spectra were measured on a Maya Pro 2000 spectrophotometer, Ocean Optics, USA. The high resolution-transmission electron microscopy (HR-TEM) (JEM- 2100, JEOL, Japan) was used to measure the morphology and size of pepsinAu NCs. Fourier transform infrared (FT-IR) spectra of pure pepsin and P-Au NCs were recorded on a FT-IR spectrometer (Shimadzu FT-IR 8400S, Japan). Zetasizer nano ZS90 (Malvern Instruments, U·K) was used to measure hydrodynamic diameter of pepsin-Au NCs. 2.3. Synthesis of P-Au NCs To remove the dissolved metals and other organic/inorganic impurities all glasswares were thoroughly washed by using aqua-regia

Pepsin is a non-specific proteolytic enzyme, which has molecular weight of ~35 kDa. It contains 327 amino-acid residues in a single polypeptide chain with three disulfide bridges. Based on the above features, the use of pepsin is explored for one-step synthesis of fluorescent P-Au NCs, which can also alter the analytical application of Au NCs towards spermine (Scheme 1). After mixing the solutions of HAuCl4 and pepsin under vigorous stirring at room temperature, the color of the solution was changed from light yellow to light brown color. This is due to the change in the oxidation state of gold from Au3+ ion to Au+ ion and then finally to Au0 atoms, which confirm that pepsin was effectively reduced the Au3+ ions to Au0 atoms to form P-Au NCs. During this reaction, the color change from light yellow to brown confirms the reduction of Au+ to Au0, and then P-Au clusters. After addition of NaOH, pH value increase up to 11.6, which improve the reducing power of protein. The absorbance and fluorescence spectra of P-Au NCs were investigated by UV–visible and fluorescence spectrometric techniques (Fig. 1). The UV–vis absorption spectrum of P-Au NCs showed a maximum absorption at 328 nm, which is quite different from the absorption spectra of pure pepsin and HAuCl4 solutions. Noticeably, there is no absorption peak in visible region (400–800 nm), revealing the larger Au nanoparticles are not formed with the use of pepsin as a reducing and capping agent. In addition, the as-synthesized P-Au NCs display a strong emission peak at 655 nm upon the excitation at 416 nm, confirming the existence of a large stoke shift (239) nm, which can effectively avoid the crosslinking between the excitation and emission peaks (Fig. 1b).

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Au3+ Au3+ Au3+ Au3+ Au3+

10 min

Au3+

Au+

Au+ Au+

Au+

Au+

HAuCl4

Pepsin

1M NaOH

3h

Application

Au0 Au NCs Histidine Glutathione Cysteine Methionine Spermidine Adenine Isatin Phenyl alanine Lysine Thymine Spermine

I/I0

1 0.8 0.6 0.4 0.2 0

Au+ Au+

λex 416 nm

λem 655 nm

Scheme 1. Schematic illustration of one-step synthetic approach for fabrication of fluorescent Au NCs using pepsin and their application in sensing of spermine.

Moreover, the obvious bright-red emission of P-Au NCs solution was easily observed with naked-eye when excited them under UV light at 365 nm (Inset of Fig. 1b). These properties clearly say that assynthesized P-Au NCs are ultra-small size (~2.0 nm), and each Au NCs contain approximately ~25 Au atoms. The quantum yield of the synthesized P-Au NCs is 7.4%, which is higher than that of other Au NCs [27,36]. To estimate the stability of P-Au NCs, the fluorescence emission spectra of P-Au NCs were measured at different time intervals i.e., from day 1 to 90 days. As shown in Supporting Information of Fig. S1, the assynthesized P-Au NCs exhibited high stability up to 45–60 days after that the fluorescence emission intensity was slightly decreased. This result confirms that the as-synthesized P-Au NCs are stable for long time, which could be more sufficient to evaluate the application of Au NCs. The HR-TEM image of P-Au NCs demonstrates that the formation of ultra-small particles with an average size of 2.0 ± 0.5 nm and well dispersed with spherical shape (Fig. 2a and b). As shown in Supporting Information of Fig. S2a, DLS data revealed the mean hydrodynamic diameter of P-Au NCs is 1.9 ± 0.5 nm. The as-synthesized P-Au NCs exhibited zeta potential at −36.7 mV (Supporting Information of Fig. S2b), indicating the as-synthesized Au NCs are well stable. FT-IR spectra of pure pepsin and P-Au NCs were shown in Supporting Information of Fig. S3. The peaks at 3286 and 2960 cm−1 correspond to stretching vibrations of \\OH,/ \\NH and \\C\\H groups of pure pepsin. The peaks at 1654 and 1548 cm−1 are ascribed due to stretching vibrations of amide band I and II. Similarly, amide-III band is observed at 1238 cm−1, while \\C\\N stretching vibration is observed at 1074 cm−1. However, these specific spectral characteristics are remarkably changed due to the binding of Au3+ ions with pepsin, thereby Au3+ ions are progressively reduced to Au0 atoms. The formed Au atoms are precisely present in the pepsin structures, favouring to form P-Au NCs with consisting of ~25

Au atoms, which yields the emission peak at 655 nm. These spectral data reveal that the formation of P-Au NCs with red emission. 3.2. Recognition ability of P-Au NCs towards spermine via fluorescence “turn-off” To explore compatibility of P-Au NCs based fluorescent probe towards biologically important -NH2 containing molecules, the fluorescence emission spectra of P-Au NCs in aqueous solution were studied with respect to various biomolecules commonly present in biofluids (histidine, glutathione, cysteine, methionine, spermidine, adenine, isatin, phenyl alanine, lysine, thymine and spermine) (Fig. 3a). Interestingly, P-Au NCs exhibited a significant degree of recognition ability towards spermine among other biomolecules, which is clearly confirmed from the emission spectra of P-Au NCs. As expected, the characteristic red emission peak at 655 nm was remarkably quenched only in the presence of spermine, whereas no fluorescence quenching was observed with other -NH2 group containing biomolecules. These results demonstrate that P-Au NCs could be a promising fluorescence turn-off sensor for spermine. 3.3. Fluorescence turn-off mechanism The formation of P-Au NCs was confirmed by various analytical techniques. The SPR band did not observe at visible region, which suggests that P-Au NCs exhibited molecular-like electronic features (HOMO-LUMO). It shows that the absorbance at ultraviolet (328 nm) regions is due to the inter-band electronic transitions from discrete energy levels in P-Au NCs. The characteristic emission peak of P-Au NCs at 655 nm was generated when excited at 416 nm, revealing the formation of fluorescent P-Au NCs, which is due to inter-band transitions. As shown

J.R. Bhamore et al. / Journal of Molecular Liquids 280 (2019) 18–24

1.0

(a)

Pepsin HAuCl4 Pepsin-Au NCs

Absorbance (a.u.)

0.8

0.6

0.4

0.2

0.0 300

400

500

600

700

800

900

Wavelength (nm)

Fluorescence intensity (a.u.)

55

(b)

Excitation Emission

50 45 40

Under day light

Under UV light at 365 nm

35 30 25 20

21

them to form non-covalent interactions (electrostatic, hydrogenbonding and van der Waal) with spermine. Since spermine exists as a polycationic structure due to the protonation of amino groups at above pH 7.4 [39]. Therefore, non-covalent interactions have generated between P-Au NCs and spermine, facilitating the aggregation of ultra-small Au NCs, which results a significant quenching of emission peak of P-Au NCs at 655 nm. To confirm this, HR-TEM image of P-Au NCs with spermine was studied (Supporting Information of Fig. S4). This result indicates that ultra-small size P-Au NCs have become into large particles due to the aggregation of P-Au NCs induced by spermine, which yields a change in color from red fluorescent to non-fluorescent. Notably, the principle of fluorescence quenching of P-Au NCs by spermine has remarkably utilized for specific and sensitive quantification of spermine in biofluids. In order to evaluate effective reaction time for quenching of fluorescence spectra, the emission spectra of P-Au NCs were studied upon the addition of spermine at different reaction time from 1.0 to 30 min (Supporting Information of Fig. S5). The time-dependent fluorescence emission spectra of P-Au NCs-spermine exhibited progressive quenching with increasing time from 1.0 to 30.0 min, revealing the fluorescence quenching was reached a plateau at 20 min. Therefore, the fluorescence emission spectra of P-Au NCs were recorded with the addition of spermine at 20 min. To evaluate the effect of pH on the sensing ability of P-Au NCs towards spermine, the fluorescence emission spectra of P-Au NCs were recorded upon the addition of spermine at phosphate-buffered saline (PBS) and Tris-HCl pH from 2.0 to 12.0 (Supporting Information of Fig. S6). These spectral studies demonstrated that no drastic quenching was observed in the presence of above buffer media pH. The spermine has shown higher quenching ability without addition of buffer pH. Thus, the fluorescence assay of spermine was carried out without addition of buffer pH.

15

3.4. P-Au NCs-based fluorescence turn-off detection of spermine

10 400

450

500

550

600

650

700

750

800

Wavelength (nm) Fig. 1. (a) UV–visible absorption spectra of pepsin, HAuCl4, and P-Au NCs solutions and (b) fluorescence excitation and emission spectra of P-Au NCs(Inset: Color of P-Au NCs solution in day light and under UV light; excited at 365 nm).

in Scheme 1 and Fig. 3a, P-Au NCs show remarkable recognition ability towards spermine, illustrating the P-Au NCs exhibit high degree of specificity for sensing of spermine. The P-Au NCs have multi-functional organic groups (\\COOH, \\NH2, \\SH, \\C_O, and \\CO\\NH\\), allowing

As shown in Fig. 3, spermine was specifically quenched the fluorescence emission spectra of P-Au NCs, which allows to develop a simple fluorescence “turn-off” mechanism for assaying of spermine. The fluorescence emission spectra of P-Au NCs at 655 nm were measured upon the addition of spermine concentration in the range of 0.0075–100 μM (Fig. 3b). The fluorescence emission peak of P-Au NCs at 655 nm was gradually quenched with increasing concentration of spermine from 0.0075 to 100 μM. As a result, a linear calibration graph between I0/I values (I0 and I represent the emission intensity of P-Au NCs at 655 nm without and with spermine) and spermine concentration in the range of 0.0075–10 μM (Supporting Information of Fig. S7). The regression curve is well fitted as the equation of y = 0.1936x + 1.3868

(a)

25

(b)

Number (%)

20 15 10 5 0 1.2

1.5

1.8

2.1

Size (nm) Fig. 2. (a) HR-TEM image of P-Au NCs and (b) histogram plotted at 10 nm scale bar.

2.4

22

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60

Au NCs

(a)

Histidine Glutathione

Table 1 Compression table for the evaluation of the present method with other analytical techniques for detection of spermine.

Fluorescence intensity (a.u.)

Cysteine

50

Methionine

Probe

Method

Linear range (μM)

Polyanionic poly(p‑phenylene ethynylene) Tyrosine-Au NPs Tyrosine-Au NPs – – ssDNA- AuNPs Organic NPs Dicarboxylated ethynylarene Pyrene‑1,3,6‑trisulfonate DNA-Au NPs CdTe QDs

Fluorescence

0–83

690

[3]

UV–visible Fluorescence HPLC/MS HPLC UV–visible Fluorescence Fluorescence Fluorescence UV–visible Fluorescence UV–visible Fluorescence Fluorescence

0.136 6.2 1 × 103 618.2 × 103 68.69 36.2 25,000 – 11.6 2.9 1.66 473.29 1000 1.75

[38] [38] [40] [41] [42] [43] [44] [45] [46] [47]

DNA-Au NPs Supramolecular complex Pepsin-Au NCs

0.0001–50 0.02–0.13 25–74 2500–105 0–2.4 – – 0–0.4 0.1–2.0 0.05–15 1–12 0–5 0–12 0.0075–10

Spermidine Adenine

40

Isatin Phenyl alanine Lysine Thymine

30

Spermine

20

1

2

3

4

5

6

7

8

9 10

12

13

10

600

630

660

690

720

750

Wavelength (nm)

Fluorescence intensity (a.u.)

50

Lod (nM)

Reference

0.0075 µM

(b)

40

30

100 µM 20 0.0075

100 µM

[48] [49] Present method

alanine, lysine and thymine, 500 μM; metal ion - Pb2+, Cu2+, Fe3+, Ni 2+ , Zn2+, Cd 2+ , As 3+ , Fe 3+ , and Al3+, 500 μM and anions - I− , Br−, Cl−, F−, PO43−, SO42−, Cr2O72− and S2−, 500 μM) has no effect on the fluorescence emission spectra of P-Au NCs. However, remarkable fluorescence quenching was observed upon the addition of spermine (100 μM) to P-Au NCs containing mixture of competitive chemical species (500 μM), signifying a typical fluorescence turn-off response of P-Au NCs only with spermine. Moreover, there is no significant variation in the fluorescence quenching by spermine in the presence and absence of mixture of other competitive chemical species, suggesting the selectivity of P-Au NCs towards spermine. 3.6. Practical application of the method

10 600

650

700

750

Wavelength (nm) Fig. 3. (a) Fluorescence emission spectra of P-Au NCs obtained when treated with -NH2 group containing biomolecules (histidine, glutathione, cysteine, methionine, spermidine, adenine, isatin, phenyl alanine, lysine, thymine and spermine, 500 μM) when excited at 416 nm (Inset: photograph of (1) Au NCs, (2) histidine, (3) glutathione, (4) cysteine, (5) methionine, (6) spermidine, (7) adenine, (8) isatin, (9) phenyl alanine, (10) lysine, (11) thymine and (12) spermine, when exposed to UV at 365 nm) and (b) sensitivity of the method was established by measuring emission spectra of P-Au NCs at 655 nm with increasing concentration of spermine from 0.0075 to 100 μM (Inset shows the changes of red fluorescence with increasing concentration of spermine).

(spermine, μM), R2 = 0.9904. The detection limit of this probe is 1.75 nM (S/N = 3), exhibiting lower detection limit than the reported other analytical- and fluorescence-based spermine assays [3,38,40–49] (Table 1). Under UV light exposure at 365 nm, the red fluorescence intensity of P-Au NCs was gradually decreased with increasing concentration of spermine, which can be distinguished with naked-eye and become dark at 100 μM of spermine (Inset of Fig. 3b). This result reveals that the sensitivity of the probe is more enough for the quantification of spermine levels in biofluids.

To validate the practical use of the probe, the proposed method was applied to detect spermine in biofluids (human plasma and urine), because spermine is recognized as a cancer biomarker, which usually varies its concentration in normal and cancer patients urine samples. The collected biofluids were spiked with different concentration of spermine (10, 50, and 100 nM) and then spermine was quantified by using P-Au NCs as a fluorescence turn-off probe. Even though there are several biomolecules, organic and inorganic chemical species found in biofluids, the probe exhibited high recovery ranges from 99.62 to 100.78% with low relative standard deviation values (0.04–0.91%), revealing the practical application of the probe for the detection of spermine in biofluids (Table 2).

Table 2 Recovery of spermine from biofluids using P-Au NCs as a fluorescent probe. Sample Plasma female

Plasma male

3.5. Interference study The spermine-specific fluorescence response of P-Au NCs was further evaluated by studying the biologically relevant chemical species (biomolecules, metal ions and anions) with and without addition of spermine. As shown in Supporting Information of Fig. S8, the presence of mixture of competitive chemical species (biomolecules - histidine, glutathione, cysteine, methionine, spermidine, adenine, isatin, phenyl

Urine female

Urine male

a

Added concentration (nM)

Found concentration (nM)a

Recovery (%)

10 50 100 10 50 100 10 50 100 10 50 100

9.97 ± 0.14 50.03 ± 0.19 100.80 ± 0.21 9.98 ± 0.05 49.94 ± 0.22 99.62 ± 0.28 9.99 ± 0.07 50.07 ± 0.13 100.78 ± 0.16 10.00 ± 0.02 50.04 ± 0.10 100.75 ± 0.35

99.79 100.06 100.08 99.89 99.88 99.62 99.93 100.15 100.78 99.94 100.09 100.75

Mean ± standard deviation (n = 3).

RSD (%) 0.22 0.44 0.54 0.11 0.50 0.73 0.14 0.28 0.43 0.04 0.23 0.91

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4. Conclusions In this work, pepsin was successfully demonstrated as a potential ligand for the reduction of Au3+ ion to Au0 and then formation of atomically precise P-Au NCs. The as-synthesized P-Au NCs are well dispersed in water and showed strong emission peak at 655 nm when excited at 416 nm. The emission peak of P-Au NCs was remarkably quenched by the addition of spermine due to either dynamic quenching or static quenching, suggesting the strong non-covalent interactions between P-Au NCs and spermine, which yields the aggregation of P-Au NCs induced by spermine. Thus, P-Au NCs acted as a nanoprobe for the fluorescence “turn-off” detection of spermine with very high selectivity among other bioactive molecular species. Importantly, P-Au NCs-based nanoprobe exhibited low detection limit of 1.75 nM, demonstrating that it could be used as a promising fluorescent sensor for the detection of spermine as a cancer biomarker in biofluids (urine and blood). The developed P-Au NCs-based nanoprobe is effectively silenced the other co-existing chemical species during the detection of spermine. Thus, the P-Au NCs probe can offer a facile and reliable analytical platform for the rapid and sensitive detection of spermine as a biomarkers in biofluids at very low volume of samples. Acknowledgements This work was financially supported by the Department of Science and Technology, Government of India under the Extra mural Research scheme of Science and Engineering Research Board (EMR/2016/ 002621/IPC). Miss. Bhamore acknowledges the Director, SVNIT, Surat for financial support under the Doctoral Program. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.molliq.2019.01.132.

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