Introduction

Transition metals are essential both biologically and environmentally [1,2,3,4]. The extreme importance of these metals arises from either their crucial roles in biochemical processes or their hazardous nature. Copper is one of these transition metals which is the third most abundant trace element in human body and exists in a variety of cells and tissues at low concentrations [5, 6]. Hence, development of new methods for selectively detection of these metal ions is highly important and demanded. A variety of methods have been reported for detection of copper such as atomic absorption spectroscopy [7,8,9], inductive coupled plasma mass spectroscopy [10, 11], flow injection [12], and voltammetry [13,14,15,16]. However, most of these methods are costly, time consuming, insufficiently sensitive and require sophisticated instruments. On the other hand, fluorescent sensors offer low costs, simplicity, and high sensitivity and selectivity in addition to real-time monitoring [17, 18]. To date, a series of fluorescent sensors have been developed for detection of copper, majority of which suffer from poor functionality in aqueous media. Efficiency of a sensor in aqueous media is a highly important factor since majority of biological processes and environmental application take place in aqueous media.

Copper(II) ions play important role in living things and its determination in biological samples, fluids, and tissues is very important since it may be used as biochemical marker for some diseases such as Wilson disease, hemochromatosis, Menkes disease, thyrotoxicosis, biliary cirrhosis, various infections, and variety of other acute, malignant, or chronic diseases (including leukemia) [19]. Wilson’s disease is an inborn defection of copper metabolism that results in the ions accumulation in several organs such as liver with progressive hepatic damage and subsequent redistribution to various extrahepatic tissues including the cornea, kidneys, and brain. The most common clinical symptoms of Wilson’s disease are symptomatic typical extrapyramidal dysfunctions and/or liver diseases. Also, this disease is an inherited disorder of copper metabolism characterized by the copper defective biliary excretion and impairment of its incorporation into ceruloplasmin [20,21,22,23,24].

Unique properties of SBA-15 as an ordered nanoporous silica material such as high specific surface area, uniform pores, thick walls, high thermal stability, and biocompatibility has led to its extensive application in surface chemistry [25], including catalysis [26, 27], drug delivery [28, 29], sensors [30,31,32,33], and separation [34,35,36,37,38]. The surface of SBA-15 needs to be modified and functionalized to take advantage of its unique properties. This may achieved by either grafting or co-condensation approaches [25]. The introduced functional groups onto the surface of SBA-15 are easily available for target species through its straight and uniform pores channels. In addition, unmodified SBA-15 is optically transparent and non-fluorescent, which makes it an excellent candidate to be used in the field of optical sensors. Furthermore, SBA-15 benefits from the precious advantage of applicability in water, which is a highly important factor for a sensor. Only a limited number of nanoporous materials have been reported as a fluorescent sensor for detection of Cu2+ ion [39, 40]. On the other hand, the SBA-15 with well-ordered cylindrical pores have attracted special interest in the biomedical field. The pore volumes and large surface areas of it allow the efficient adsorption of a wide range of molecules, including therapeutic proteins [41, 42], small drugs [43, 44], antibodies [45], and antibiotics [46, 47]. Accordingly, these structures have been proposed for use as potential vehicles for controlled delivery of multiple therapeutic agents, real-time diagnosis, and biomedical imaging.

In this paper, we have reported a fluorescence sensor based on SBA-15 for detection of Cu2+ in aqueous media. It can be efficiently used for determination of Cu2+ in blood samples. The feasibility of the strategy for the fabrication of optical sensor for the detections of Cu2+ ions in blood was demonstrated by the high sensitivity and selectivity of the sensor. This sensor is inhibited by I¯ as masking agent and to the best of our knowledge, this is the first fluorescence sensor for determination of Cu2+ with I¯ as an inhibiting agent.

Experimental

Materials

All solvents and chemicals were commercial reagent grade and used as received from Merck and Aldrich. The details of the used chemicals along with instrumentation, data are available in Electronic Supplementary Material (ESM).

Instrumentation

X’Pert Pro MPD diffractometer was utilized for low-angle XRD measurements. N2 adsorption–desorption isotherms were recorded using a BELSORP-mini II instrument. The surface images were taken with TESCAN (MIRA3) FESEM. TEM images were obtained on a Leo 912 AB electron microscope. EDS analysis was obtained with a Philips Tecnai 20 transmission electron microscope. TGA curves were recorded using a TGA Q50 V6.3 Build 189 instrument. AWQF-510A FT-IR spectrometer and KBr disks were used to obtain FT-IR spectra. PL spectra were recorded on a Cary Eclipse fluorescent spectrophotometer. The operational details are available in the ESM.

Synthesis of sensor

The studied sensor was prepared through modification of the surface of SBA-15 channels by piperazinyl propyl methyl dimethoxysilane, which provided potentially reactive organic groups on the surface for further functionalization steps. In subsequent, the piperazinyl groups on the surface of SBA-15 channels were reacted with iminostilbene carbonyl chloride to obtain the final product. The preparation steps are illustrated in Scheme I and more details are shown in the ESM.

Scheme I
scheme 1

Schematic procedure for preparation of SBA-PI

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The procedure of detection of Cu2+ by sensor

The procedure for the selective detection of Cu2+ ions in water was performed as follows. At first step, different concentrations of Cu2+ solution were added to a dispersed SBA-PI in water and were analyzed. In the second step, the control tests for investigation of the interference effect of foreign ions Na+, Co2+, K+, Cu2+, Mn2+, Ni2+, Cd2+, Fe2+, Fe3+, Zn2+, Pb2+, Al3+, Ca2+, Cr3+, Hg2+, and Mg2+ were correspondingly conducted. Finally, by following the same above procedure, the developed optical fluorescence sensor was applied in the detection of Cu2+ ions fresh human blood samples (the normal human blood samples were kindly provided by the local hospital). To prepare the real sample, to a 1 mL of fresh blood, the appropriate amount of sodium citrate as coagulant agent was added, and the resultant mixture was centrifugated for 5 min at 2500 rpm. The supernatant or plasma phase was collected and a 100 μL portion was transferred to a spiked solution contains 2.0, 10, 50, 250, 500, 1000, 2000, and 2500 nM of Cu2+.

The quenching efficiencies of by metal ions toward SBA-PI were calculated according to the following equation:

$$ \mathrm{Quenching}\ \mathrm{efficiencies}=\frac{\left( Fo-F\right)}{Fo} $$

where F0 and F refer to the fluorescence intensities of SBA-PI (λem 309 nm) in the absence and presence of metal ions, respectively. Additionally, the developed method was compared to the classical atomic absorption spectrometry (AAS) Cu2+ ions in real blood samples in the clinical laboratory.

Results and discussion

Characterization

According to FESEM images, the prepared materials are rod-like (Fig. 1, right) and the TEM images showed the uniform channels of SBA-15 (Fig. 1, left), and the images have revealed that the channels are open in the direction of the mesoporous particles. The length and width of rods were about 5 μm and 500 nm, respectively. In addition, it was found that the prepared meso-channels (2D-hexagonal) were highly ordered along the length of the rods and the structure of the nanomaterials remained intact during surface modification.

Fig. 1
figure 1

TEM image (left) and FESEM image (right) of SBA-PI

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EDS analysis was carried out to determine the elemental composition of the SBA-PI nano materials (Fig. 2 (i)). The obtained results confirmed the presence of carbon, silicon, nitrogen, and oxygen elements in the final product, which can be taken into account as a proof of existence of the synthesized structures.

Fig. 2
figure 2

(i) EDS spectra of SBA-PI structure. (ii) N2 adsorption–desorption isotherms for SBA-15 and SBA-PI (inset: textural properties of SBA-15 and SBA-PI). (iii) TGA curves for SBA-P and SBA-PI. (iv) Low-angle XRD patterns of SBA-15 and SBA-PI. (v) FT-IR spectra of SBA-15, SBA-P, and SBA-PI

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The surface physical properties of the synthesized SBA-15 was further investigated by N2 adsorption–desorption isotherms. Porosity of the SBA-15 was measured before and after modification steps. N2 adsorption–desorption isotherms obtained for both SBA-15 and SBA-PI (Fig. 2 (ii)) demonstrated type IV standard IUPAC isotherms corresponding to mesoporous materials. At relatively high pressures (P/P0 of 0.6–0.8), a H1 hysteresis loop was observed in the isotherm of SBA-15 which it can be assigned to the sharp capillary condensation occurring within the uniform pore channels. These characteristics are attributed to ordered mesoporous materials. A similar isotherm was obtained for SBA-PI with relatively lower amount of gas adsorption and lower hysteresis loop, which it is in consistent with the shrinkage of pore volume (V), specific surface area (SBET), and average pore diameter (D) (Fig. 2 (ii), inset). This phenomenon was happened due to the introduction of the organic moieties into the pore walls of SBA-15. While the observed hysteresis loop in the isotherm of the final product verified the previously discussed results obtained from XRD analysis, it also suggested the preservation of the mesostructure. It furthermore indicated that the pore channels of SBA-15 were still available after attachment of the organic moieties onto the pore walls for later applications.

Thermogravimetric analysis (TGA) was performed for both SBA-P and SBA-PI (Fig. 2 (iii)) to estimate the amount of the grafted organic moieties onto the pore walls of SBA-15. An initial weight loss was observed in the TGA curves for both SBA-P and SBA-PI up to 200 °C. This may be assigned to removal of the physically absorbed water as well as other volatiles in the pore channels of SBA-15. Subsequent major weight losses observed for both materials were attributed to decomposition of the piperazinyl propyl groups and the grafted iminostilbene groups (about 27%). According to the numerical values indicated in the TGA curves, the amounts of the organic groups attached onto the pore walls of SBA-15 were estimated as 0.79 mmol g−1.

An intense reflection at about 2θ = 1° diffracted from (100) plane was observed in the low-angle powder X-ray patterns of SBA-15 and SBA-PI (Fig. 2 (iv)) as well as two other reflections diffracted from (110) and (200) planes, which confirmed the long-range periodic order of SBA-15 and two-dimensional hexagonal mesostructure of the material with p6mm space group [48]. The same reflections were observed in the pattern of SBA-PI, which indicated the conservancy of the SBA-15 structure after two functionalization reaction steps. Intensities of the reflections in the XRD pattern of the final product was relatively lower than those of the SBA-15, which is attributed to the introduction of organic groups onto the pore walls of SBA-15 resulting in partially lower long-range order.

FT-IR spectra of SBA-15, SBA-P, and SBA-PI were recorded for further verification of the attachment of the organic moieties onto the pore walls of SBA-15 (Fig. 2 (v)). The strong bands observed at around 800 and 1077 cm−1 in all three spectra are attributed to Si-O-Si stretching vibrations. The bands observed at 2860–2970 cm−1 in the spectrum of SBA-P and SBA-PI are assigned to symmetric and asymmetric stretching vibrations of -CH2-, which confirmed the attachment of propyl groups into the channels of SBA-15. Appearance of new bands at 1498 and 1558 cm−1 can be assigned to the aromatic C=C stretching vibrations. In addition, the band observed in the spectrum of SBA-PI in the 1783 cm−1 confirmed the attachment of iminostilbene groups on the walls of the modified SBA-15 and it is related to carbonyl groups. In overall, the three FT-IR spectral profiles are in consistent with previous characterization results and confirmed that the fluorophore groups have been successfully grafted onto the pore walls of SBA-15 channels.

Study of the sensing ability of SBA-PI

To evaluate the fluorescence ability of SBA-PI as a fluorescence sensor, water suspension of free SBA-PI was excited at the wavelength 270 nm and an intense fluorescence emission band was observed with a maximum around 309 nm. The changing of the of the emission intensity was investigated upon addition of different metal ions including Na+, Co2+, K+, Cu2+, Mn2+, Ni2+, Cd2+, Fe2+, Fe3+, Zn2+, Pb2+, Al3+, Ca2+, Cr3+, Hg2+, and Mg2+ to the water suspension of free SBA-PI. At first, fluorescence spectrum of 0.2 gL−1 water suspension of free SBA-PI (3 mL) was recorded. Then, 100 μL of 1 × 10−2 M solutions of the mentioned metal ions was separately added to the same volume (3 mL) of 0.2 gL−1 water suspension of SBA-PI, and their spectra were recorded. The obtained results showed that fluorescence emission of SBA-PI quenched remarkably in the presence of Cu2+ ion whereas the other metal ions negligibly affected the emission band intensity (Fig. 3). Therefore, SBA-PI showed to be a potential selective sensor for detection of Cu2+ ion in aqueous solutions.

Fig. 3
figure 3

Fluorescence emission spectra of SBA-PI (3 mL H2O suspension, 0.2 gL−1) in the presence of a variety of metal ions (100 μL, 1 × 10−2 M)

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Study of pH effect

In addition to the impact of pH of solution on the common form of the ions in the aqueous solutions, it can change the protonation degree of the effective functional groups in the heterogeneous optical sensor surface. For this purpose, pH of dispersed sensors was varied in the range 2.5–8.5 with acetate buffer solutions and the fluorescence response of SBA-PI was studied in the presence and absence of Cu2+ ions. In the acidic pH values, response of the sensor decreased, perhaps due to the interaction of H+ into the SBA-PI surface at strong acidic condition that induces the nitrogen atom protonation and block its reaction. In the high pH values, the fluorescence intensity increased because of the deprotonation of the functional group. In addition, the copper ions were precipitated and converted to Cu(OH)2 under strong alkaline condition which made them ineffective. In the pH range 4.0–7.0, acidity does not have any observable effects in the detection of Cu2+ions with the fabricated optical sensor shown in Fig. 4. The prepared mesoporous material can be used at pH 6 and all subsequent experiments buffered with (NaOAc–HOAc) solution. These experiments showed that SBA-PI has very good potential for the Cu2+ ions detection in real samples and biological media.

Fig. 4
figure 4

pH effect on the fluorescence intensity on the free SBA-PI and in the presence of Cu2+ ions

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Stern-Volmer plot

The quenching mechanism of SBA-PI in the presence of Cu2+ was further investigated by fluorometric titration data and corresponding plot according to Stern-Volmer Eq. (1):

$$ {F}_0/F=1+{\mathrm{K}}_{\mathrm{sv}}\left[\mathrm{Q}\right] $$
(1)

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. [Q] is the concentration of the quencher, i.e., Cu2+ and Ksv are the quenching constant. Plotting of the F0/F vs. concentration of Cu2+ ion results in a linear plot (inset of Fig. 5, left). The linear plot indicated that the quenching mechanism is either purely static or dynamic. To determine the dominant mechanism, absorption spectra of SBA-PI were recorded in the absence and presence of Cu2+ ion. The absorption spectrum of SBA-PI in the presence of Cu2+ ion 1.5 × 10−4 M was distorted from that of free SBA-PI (Fig. 5) implying that a complex was formed between the sensor and the target ion in the ground state (Fig. 5, right). The difference between free and complexed fluorophore absorption spectra suggested the highly likely of the existence static quenching. The resulted quenching constant was calculated 2.1 × 105 mol−1 by using the Stern-Volmer equation.

Fig. 5
figure 5

Left: absorption spectra of free SBA-PI (a) and SBA-PI + Cu2+ (b) (inset: Stern-Volmer plot of SBA-PI in the presence of Cu2+ ion). Right: suggested interaction of SBA-PI with Cu2+ ion

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Titration of SBA-PI by Cu2+ ion

The fluorescence intensity of SBA-PI was studied as a function of the concentration of Cu2+ ions. Upon stepwise addition of a Cu2+ solution to a 3 mL water suspension of SBA-PI (0.2 gL−1), a monotonic reduction of the emission intensity was observed (Fig. 6). A linear relationship between emission intensity and concentration was obtained (inset of Fig. 6). Furthermore, the detection limit was estimated as 8.7 × 10−9 M on the basis of DL = 3σ/m (σ = standard deviation of intercept from calibration curve and m = slope of the calibration curve), which reveals high sensitivity of the sensor toward Cu2+ ion.

Fig. 6
figure 6

Titration of SBA-PI (3 mL H2O suspension, 0.2 gL−1) by Cu2+ ion (1, 2, 3, …, 20 × 10−5 M), (inset: fluorescence emission of SBA-PI as a function of Cu2+ concentration)

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Selectivity study of SBA-PI for Cu2+

To evaluate the efficiency of the sensor in detection of Cu2+ in multi-analyte environments, a selectivity experiment was performed. The fluorescence emission of the sensor was monitored in the presence of the specified amount of Cu2+ and different amounts (200–1000 times higher than Cu2+ ions) each of the common interfering metal ions. The selectivity results, shown in Fig. 7 and Table 1, revealed that competing ions did not interfere in detection of Cu2+ ion by the sensor and the presence of studied ions has no substantial effect on the sensing process of Cu2+ ions and also it was found that the selectivity of the proposed technique reaches a desirable level for the sensing of Cu2+ ions in the complicated matrices as a real sample. With respect to the obtained results in the selectivity experiments, SBA-PI can be considered as a selective sensor for detection of Cu2+ ion.

Fig. 7
figure 7

Selectivity of SBA-PI (3 mL H2O suspension, 0.2 g L−1) for Cu2+ (100 μL, 1 × 10−2 M) in the presence of equal amounts of various metal ions

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Table 1 The effect of interfering ions on the quenching efficiency of Cu2+ ions
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Masking agent mechanism

It was observed that the quenched emission of the sensor by Cu2+ ion can be restored by the addition of I¯ anion to the medium, which is probably due to oxidation of I¯ and reduction of Cu2+ and consequent formation of CuI (Ksp = 1.1 × 10−12) which resulted and consequent formation of CuI which resulted in decreasing the interaction of Cu2+ ions with the sensor.

Comparison with other optical sensors

A comparison of the limit of detection for the studied sensor with several other materials reported in the literature in the detection of Cu2+ ions with photoluminescence determination technique has been carried out and the results are shown in Table 2. The results achieved showed that the SBA-PI has significant lower limit of detection with respect to the other prepared compounds. The mesoporous sensors have a high surface area, which makes them suitable media in comparison to the non-porous structures in ions detection.

Table 2 Comparison of the SBA-PI sensor with other reported materials for detection of Cu2+
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Fluorimetric analysis of Cu2+ in spiked blood samples

The feasibility of application of the SBA-PI-based optical sensor was investigated in analyzing Cu2+ concentrations in different spiked blood samples. A linear relationship in the range 2.0–2500 nM (R2 = 0.9842), between the quenched fluorescence and the logarithm of the concentrations of Cu2+ ions (Fig. 8 (i)) was obtained. The validity of the obtained results by the fluorescence sensor was checked by correlation analysis and comparison with calculated Cu2+ ions in blood by AAS technique in clinical laboratory by detecting Cu2+ ions in real blood samples of R2 = 0.9875 (P < 0.050) (Fig. 8 (ii)). Obviously, there is no significant difference between obtained results by two methods in analyzing Cu2+ ions in measured blood. Furthermore, the stability of SBA-PI stored in the fresh blood was investigated (Fig. 8 (iii)), showing no significant change of fluorescence intensity up to 7 days. Hence, the developed optical sensor with SBA-PI as the fluorescent sensors can promise the potential of serving as a reliable and rapid candidate for the sensitive and selective detections of Cu2+ ions, where mentioned sensors with powerful fluorescence might circumvent the problems of the interferences from the scattering and absorption effects of protein backgrounds in human blood.

Fig. 8
figure 8

(i) Fluorescence quenching efficiencies of SBA-15 versus the logarithmic concentrations of Cu2+ ions (2.0, 10, 50, 250, 500, 1000, 2000, 2500 nM) separately spiked in blood samples. (ii) The correlation of the detection results of Cu2+ ions in real blood samples between the developed optical sensor method and the classic AAS method. (iii) The stability of SBA-15 stored in blood over different time intervals

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Conclusion

In summary, a fluorescent sensor based on SBA-15 was synthesized and then characterized, successfully. FESEM, TEM, EDS, low-angle XRD, N2 absorption–desorption isotherms, TGA, and FT-IR analysis illustrated that SBA-15 was synthesized and the organic groups were attached onto the pore walls of mesoporous channels, successfully. Fluorescence studies showed that Cu2+ ion quenched the emission of SBA-PI while the rest of the tested metal ions barely influenced the emission. Selectivity of the sensor toward Cu2+ ion was verified in the presence of a variety of common competing metal ions. Titration studies demonstrated that the fluorescence emission intensity of the sensor was a function of the concentration of the target ion and decreased gradually upon incremental addition of Cu2+ ion. Furthermore, the sensor proved to work efficiently in real sample waters. With the unique modified SBA-15 were tailored for a fluorescence quenching-based analysis method for probing Cu2+ ions in human blood. In addition, the reversible Cu2+ analysis can be expected by restoring the fluorescence of alloying SBA-PI after the tests. Also, the SBA-PI sensor can ensure the optical sensor analysis with the minimized interferences from the protein backgrounds in blood. On the other hand, studying the logic behavior of the sensor showed, SBA-PI can act as an INHIBIT-type logic gate with Cu2+ and I¯ as the inputs. The fabricated SBA-PI-based optical sensor strategy is simple, rapid, selective, highly sensitive, and has great promising for the selective detections of Cu2+ ions in the clinical, food hygiene, and environmental monitoring fields. The sensitivity and low detection limit of the sensor enable it as very good device in monitoring Cu2+ in patients’ blood suffered from Wilson’s disease.