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A label-free G-quadruplex-based fluorescence assay for sensitive detection of alkaline phosphatase with the assistance of Cu2+
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 227 (2020) 117607
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A label-free G-quadruplex-based fluorescence assay for sensitive detection of alkaline phosphatase with the assistance of Cu2þ Lin Ma, Xue Han, Lian Xia, Fengli Qu, Rong-Mei Kong* College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu Shandong, 273165, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2019 Received in revised form 7 August 2019 Accepted 6 October 2019 Available online 7 October 2019
The level of alkaline phosphate (ALP) is a significant biomarker index in organism. In this work, a labelfree and sensitive G-quadruplex fluorescence assay for monitoring ALP activity has been developed with the assistance of Cu2þ based on the competitive binding effect between pyrophosphate (PPi) and Gquadruplex-N-methylmesoporphyrin (G4/NMM) complex to Cu2þ. In the sensing assay, the G4/NMM complex is employed as a signal indicator, while the Cu2þ as a quencher and the PPi as recovery agent as well as the hydrolytic substance for ALP. In details, the fluorescence of the G4/NMM complex was efficiently quenched by introducing Cu2þ due to the proximal carboxylate groups of NMM coordinating with the Cu2þ as well as the unfolding of G-quadruplex by Cu2þ, while the higher affinity between PPi and Cu2þ could lead to the fluorescence recovery. However, in the presence of ALP, the PPi was hydrolyzed to phosphate ions (Pi) which cannot integrate with Cu2þ, resulting in the fluorescence quenching once again. Thus, a simple and facile way to inspect ALP has been exploited. The proposed assay shows a good linear relationship in the range from 0.5 to 100 U/L with the detection limit of 0.3 U/L. Moreover, the fabricated method is succeeded in detecting ALP in human serum samples, indicating the potential as a profitable candidate in biological and biomedical application. © 2019 Elsevier B.V. All rights reserved.
Keywords: G-quadruplex Label-free Alkaline phosphatase Cu2þ Fluorescence
1. Introduction Alkaline phosphate (ALP) is an essential metabolic enzyme with the function of catalyzing the dephosphorylation process [1,2]. As a widely distributed membrane-bound enzyme, ALP has broad substrate specificity that is able to hydrolyze a wide variety of phosphated compounds in vivo and in vitro. It is participated the dephosphorylation process of a variety of biomolecules, including nucleic acids, proteins and small molecules [3]. In another way, ALP is an indispensable biomarker in diseases’ diagnose. It is present widely in living tissues especially in liver, kidneys, prostate and bones. In the cycle of growth and apoptosis, ALP plays a key role in signal transduction and intracellular regulation [4]. The abnormal level of ALP in serum has been verified to be closely related to various diseases including diabetes, prostate cancer, bones disease and liver dysfunction [5e8]. Therefore, there is an urgent need that a sensitive and selective method should be developed to monitor ALP level. Up to now, a variety of facile strategies have been reported to determine of ALP level containing of colorimetry [9,10], surfaceenhanced Raman scattering [11], electrochemistry [12,13],
* Corresponding author. E-mail address: [email protected] (R.-M. Kong). https://doi.org/10.1016/j.saa.2019.117607 1386-1425/© 2019 Elsevier B.V. All rights reserved.
chemiluminescence [14] and fluorescence methods [15e18]. Compared to the traditional methods that have the defects of time-consuming and complicated operation, the fluorescence strategies for the detection of ALP have drawn wide attention because of the inherent merits such as higher sensitivity, easier devise and less modification. Many fluorescence assays have been done to explore high performance methods for the detection of ALP. For example, organic fluorophores [19,20], semiconductor quantum dots and metal nanoclusters [21e23] have been successfully applied to monitor ALP activity. However, most of these mentioned methods suffer from several limits such as low biocompatibility, costly reagents and complex synthesis routes. Therefore, develop simple, cost effective and more biocompatible fluorescent strategy for evaluating ALP level is still highly needed. Since ALP has broad substrate specificity, several substrates including different original or designed substrates have been employed as the hydrolyzed substances. There were adenosine triphosphate (ATP), pyrophosphate (PPi), p-nitrophenyl phosphate (PNPP), 4-methylumbellyferyl phosphate, 2-phosphate-L-ascorbic acid (AAP) and so on [24e26]. Therefore, by the catalyzing of the substrates hydrolysis, great progress has been made in developing sensitive and selective fluorescence methods for monitoring of ALP activity. Among many nature substrates for ALP, PPi has attracted much more attention due to its unique properties that it has a lower
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pH optimum compared to other substrates [27] and strongly chelate action with certain metal ions such as Cu2þ, Hg2þ and Zn2þ [28]. Traditional methods using radiolabeled PPi as ALP substrates were laborious with high detect limits rather than taking use of label-free substrate. Nowadays, various efforts were taking usage of label-free PPi to design ALP assays. For example, based on the inhibition of dsDNA-templated copper nanoparticles by PPi, Zhang et al. designed a label-free fluorescent strategy for the detection of ALP activity [23]. Liu et al. synthesized near-infrared fluoresence tryptophan-functionalized CuInS2 quantum dots (WeCuInS2 QDs) and used for the detection of ALP based on the quenching effect of ALP hydrolysis of PPi induced release of Cu2þ [29]. Although the mentioned fluorescence methods were effective for the detection of ALP, they are still suffering from the limits of complicated nanomertial synthesis procedure and the complex post-treatment process. Therefore, it is necessary to develop new simple and reliable methods for the determination of ALP with high sensitivity and selectivity. G-quadruplex is a structure which consists of stacking of planar G-tetrads [30]. Several small dye molecules combines with Gquadruplex can greatly enhanced the autologous fluorescence. Serving as the fluorescent signal output, G-quadruplex/dye molecule (G4/dye) complexes have been applied for the construction of multifarious biosensors with high biocompatibility. For example, zinc protoporphyrin IX (ZnPPIX), thioflavin T (ThT) and N-methylmesoporphyrin (NMM) are frequently employed as label-free G4/ dye molecule probes in detecting metal ions, DNA and small molecules [31e36]. Among these small dye molecules, NMM not lony is a commercially porphyrin derivative that has a pronounced structural selectivity for G-quadruplex with the increase of fluorescence rather than duplex, triplexes, double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), but also emissions fluorescence at the wavelength longer than 600 nm [37,38]. In such a case, when applied in biological system, the long wavelength fluorescence emission of the G4/NMM complex enables it to resist the interference of autofluorescence. In addition, the fluorescence of G4/NMM can be quenched by Cu2þ via the proximal carboxylate groups of NMM combining with Cu2þ as well as the unfolding of G-quadruplex by Cu2þ [39]. Inspired by the fact that the G4/NMM needs no more modification and can be simple manipulated, herein, a label-free fluorescence assay was established to monitor ALP level based on the quenching and recovery of the fluorescence of G4/NMM with the assistance of Cu2þ. The PPi was used for the inhibition of the quenching effect of Cu2þ to the fluorescence of G4/NMM due to the higher chelate affinity between PPi and Cu2þ. However, the hydrolyzation of PPi to phosphate ions (Pi) due to the presence of ALP resulted in the releasing of Cu2þ. The fluorescence of G4/NMM was requenched by the released free Cu2þ and the concentration of ALP was related to the fluorescence changes. Therefore, utilizing Gquadruplex without any modification, a type of label-free “off-onoff” fluorescence method was established for sensitive mornitoring of ALP activity. The assay was also applied for the detection of ALP in human serum samples with satisfactory results. Compared to the known nanomaterials based and molecular sensors for ALP reported in the literatures, our proposed approach possesses some remarkable features: (1) The DNA strands do not require any labeling, leading to less laborious and more cost-effective synthesis; (2) the commercially available NMM not only requires no complex synthesis but also emissions fluorescence at the wavelength longer than 600 nm when combined with G4. The red fluorescence emission feature enables it to resist the interference of autofluorescence when applied to biological sample analysis; and (3) the high sensitivity and specificity can be achieved via a simple “mix and detection” procedure, which are comparable or superior to the reported methods.
2. Experiments section 2.1. Reagents The oligonucleotide G4 (50 -GGGTTGGGCGGGATGGGG-30 , purified by HPLC) was synthesized and purchased from Takara Biotechnology Co. Ltd. (Dalian, China). N-methylmesoporphyrin (NMM, 96%), pyrophosphate (PPi, 99%) and alkaline phosphatase (ALP, lyophilized powder) were purchased from Beijing J&K Scientific Co., Ltd. (Beijing, China). The CuSO4$5H2O was purchased from Sinopharm Chemical Reagent Beijing Co. Ltd. Glucose (Glu), glucose oxidase (GOx), bovine serum albumin (BSA), pepsin, thrombin and immunoglobulin G (IgG) were purchased from Sigma-Aldrich. Prostatic specific antigen (PSA) was purchased from Shanghai Linc Bio Science Co. Ltd. Immunoglobulin G (IgG) was purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. The buffer used in this experiment was 25 mM HEPES buffer (pH 7.4), which was prepared by HEPES and HCl containing 100 mM NaCl, 10 mM KCl and 2.5 mM MgCl2$6H2O. All of the chemical reagents were analytical grade without any purification. The water used throughout all experiments was purified through a Millipore system (resistivity>18 MU cm). 2.2. Apparatus All the fluorescent spectrums were performed by F-7000 spectrometer (Hitachi, Japan). The experiment settings were as following: excitation wavelength was at 399 nm using 10/10 nm slit width, PMT detector voltage was 700 V. The emission spectra were recorded within the range of 580-700 nm. The pH measurement was carried out on a Mettler-Toledo Delta 320 pH meter at room temperature (about 25 C). 2.3. Preparation of G4/NMM Before the experiment, the G4 oligonucleotide was heated at 95 C for 5 min and then cooled down at 4 C in the refrigerator. G4/ NMM was achieved by diluting the stock solution of G4 and NMM with 25 mM HEPES buffer. The mixture was incubated at 37 C for 30 min to allow the formation of G4/NMM. 2.4. Fluorescence detection of PPi To verify the inhibition of the PPi to the quenching effect of Cu2þ, the fluoresence of G4/NMM was investigated in the presence of different concentrations of PPi. First, 10 mL of 100 mM Cu2þ was premixed with 10 mL of PPi with different concentrations. Then, the Cu2þ-PPi complexs were introduced to the above prepared G4/ NMM solutions and incubated for 25 min at room temperature. Finally, the fluorescence spectra of these solutions were recorded in the wavelength rang of 580-700 nm. 2.5. Fluorescence detection of ALP Fluorescence detection of ALP was conducted in 500 mL Eppendorf tubes. 5 mL of ALP with various concentrations and 5 mL of PPi (1.2 mM) were premixed at 37 C for 30 min and then 10 mL of Cu2þ (100 mM) was added into the mixture at room temperature. Finally, the mixture was added to the solution that containing 20 mL of G4/ NMM and 160 mL HEPES buffer, and incubated at room temperature for 25 min. The fluorescence spectra of the mixture were recorded in the wavelength rang of 580-700 nm. The real complex samples for detecting ALP were conducted in diluted human serum samples, and the detection procedure was similar to the above experiments in buffer solution just containing 1% diluted human serum including 5 mM Cu2þ and 30 mM PPi. Serum sample experiments
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were performed according to the Guidelines for Ethical Committee, Qufu Normal University. All studies were approved by Ethical Committee, Qufu Normal University. Informed consents were obtained from human participants of this study. 3. Results and discussion 3.1. Mechanism of the assay for ALP detection The principle of the designed strategy to detect ALP based on label-free G-quadruplex and competing binding reaction with the mediate of the Cu2þ was depicted in Scheme 1. As is known to all, the fluorescence of some dye molecules such as NMM can be extremely enhanced by combining with G-quadruplex to form complex [37]. In addition, the fluorescence of G4/NMM can be quenched by Cu2þ via the proximal carboxylate groups of NMM combining with Cu2þ as well as the unfolding of G4 by Cu2þ, with the association constant (Ka) of NMM with Cu2þ is 1.82 106 M-1 [39]. Inspired of the point, a label-free assay was developed for the detection of ALP based on the inhibition of PPi to the quenching effect of Cu2þ to G4/NMM complex. In details, the G4/NMM complex fluorescence could be quenched due to the proximal carboxylate groups coordinating with the Cu2þ to form Cu2þ/NMM. In another way, Cu2þ caused the unfolding of G-quadruplex as well as induced the fluorescence quenching of G4/NMM complex. The higher association between PPi and Cu2þ (1.0 109 M-1) disturbed the interaction between Cu2þ and G4/NMM, thus leading to the fluorescence recovering. The introduction of the ALP would hydrolyze PPi into Pi so that the Cu2þ was released into solution. The free Cu2þ combined with G4/NMM complex, which resulted in the fluorescence quenching again. As a whole, in the presence of Cu2þ and PPi, the fluorescence was recovered, while the introduction of ALP induced the fluorescence quenching reoccurs. Since the fluorescence quenching of the G4/NMM complex was related to the concentration of ALP, the proposed principle is effective to monitor ALP level. Meanwhile, no complicated synthesis procedure and modification make the proposed label-free assay simple and costeffective. 3.2. Feasibility of the designed strategy As a proof of concept to evaluate the feasibility of our design for ALP detection, the fluorescence experiments were carried out
Scheme 1. Schematic illustration of the label-free detection strategy for ALP based on the G4/NMM complex with the assistance of Cu2þ and PPi.
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under different conditions. As is shown in the Fig. 1, the G4/NMM complex exhibited strong fluorescence at 610 nm (curve a). A significant quenching of the G4/NMM fluorescence can be observed when the Cu2þ was added into the solution (curve b). Furthermore, after the addition of PPi, the fluorescence was obviously recovered (curve c). The results show that the G4/NMM complex exhibited an obvious “off-on” phenomenon with the assistance of Cu2þ and PPi. However, with a certain amount of ALP was introduced into the mixture including G4/NMM, Cu2þ and PPi, one can obvious observe the fluorescence “on-off” phenomenon (curve d) due to hydrolyzation of PPi to Pi by ALP induced releasing of free Cu2þ. Therefore, the results indicate that the proposed label-free can be used for the effective detection of ALP.
3.3. Optimization of detection conditions To achieve the best analytical performance of the proposed assay for ALP detection, some detection parameters involved in this assay were optimized. Since the fluorescence enhancement was directly related to the increasing concentration of G-quadruplex, the fluorescence experiment was first carried out in the presence of NMM at a fixed concentration of 0.3 mM. As shown in Fig. 2, with the increase of G4 concentrations range from 0 to 0.4 mM, the fluorescence intensity increases gradually and can reach the plateau at the G4 concentration of 0.3 mM, indicating the complete binding of NMM by G4. It is obvious to know that the redundant G4 cannot cause the further fluorescence increasing, which may be due to the binding concentration ratio between NMM and Gquadruplex is 1:1 [39]. For the phenomenon, 0.3 mM G4 was chosen for the further experiments. Then, the related experiment conditions of the G4/NMM fluorescent sensing platform for the detection of ALP activity with the assistance of Cu2þ were optimized. Quantative detection of ALP was based the “off-on-off” model of the G4/NMM fluorescence. Therefore, the optimal concentration of Cu2þ used to quench the fluorescence of G4/NMM was investigated. As shown in Fig. 3A, with the increase of Cu2þ concentration, the fluorescence of G4/NMM at 610 nm was decreased gradually due to the coordination of NMM with Cu2þ as well as the unfolding of G-quadruplex by Cu2þ. The quenching efficiency could even achieve as high as 89% under the concentration of 3 mM Cu2þ. The time dependence of the fluorescence after the addition of Cu2þ was studied due to that the fluorescence quenching was significantly related with the binding time between Cu2þ and NMM. The Fig. 3B shows that the fluorescence quenching reaches the plateau when the incubation time reaches
Fig. 1. Fluorescence spectra under different conditions: (a) G4/NMM, (b) G4/ NMM þ Cu2þ, (c) G4/NMM þ Cu2þ þ PPi, (d) G4/NMM þ Cu2þ þ PPi þ ALP. The final concentrations of G4/NMM, Cu2þ, PPi and ALP are 0.1 mM, 5 mM, 30 mM and 100 U/L, respectively.
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enzymatic reaction time of ALP for the hydrolysis of PPi was also very important for the G4/NMM complex based sensing system. According to the experiment results shown in Fig. 3D, the catalytic hydrolysis of PPi can be completed within 30 min.
3.4. Analytical performance of sensing strategy for ALP detection
Fig. 2. Fluorescence intensities of G4/NMM at 610 nm. The final concentration of NMM is 0.3 mM. Error bars were estimated from three replicate measurements.
25 min, implying the appropriate time of Cu2þ quenching was 25 min in the designed system. Thus, 3 mM Cu2þ and 25 min incubation time of Cu2þ are choosen for the following experiments. Next, the optimized Cu2þ concentration was used to investigate the fluorescence changes of the G4/NMM complex by adding Cu2þPPi complex with the PPi concentrations range from 0 to 50 mM. As shown in Fig. 3C, the fluorescence intensity gradually increases with the addition of PPi from 0 to 30 mM, and then kept almost unchanged, indicating that the fluorescence recovery proceeded in a dose dependent response to PPi. The luorescence recovery could be reached a zenith at PPi concentration of 30 mM, which was employed in the subsequent experiments. Additionally, the
To testify the ability of the Cu2þ-mediated G4/NMM assay for ALP detection, various different concentration of ALP under optimized conditions was investigated. Fig. 4A depicts the fluorescence spectra of G4/NMM sensing system for the detection of ALP with the concentrations rang from 0 to 200 U/L. It is clearly observed that the peak intensity decreased gradually with increasing of ALP concentration. As shown in Fig. 4B, the plot of the peak intensity of G4/NMM complex shows an excellent linear correlation versus the ALP concentrations range from 0.5 to 100 U/L. The calibration equation was DF ¼ 3.08 þ 7.03[ALP] (R2 ¼ 0.995), where DF is the fluorescence decrease intensity of G4/NMM at 610 nm between the absence and presence of ALP with different concentrations, [ALP] is the concentration of ALP. A detection limit of 0.3 U/L can be obtained from the calibration curve according to the definition of detection limit (3s/slope). The sensitivity is enough for ALP detection in biological samples as the normal range of serum ALP in adults is about 46-190 U/L [40]. In addition, the analytical performance of proposed assay was compared to the previously reported for ALP in Table S1. The proposed biosensor revealed superior characteristic in wide linear range and low detection limit. The results indicated the proposed label-free G4/NMM complex based sengsing system has the potential to be extended to practical fluorescence detection of ALP.
Fig. 3. (A) The effect of the amount of Cu2þ on the fluorescence intensity of G4/NMM complex. (B) The effect of the incubation time of Cu2þ with G4/NMM on the fluorescence intensity of G4/NMM complex. (C) The effect of the amount of Cu2þ-PPi complex with a fixed Cu2þ concentration at 3 mM and different amounts of PPi. (D) The effect of the enzymatic reaction time of ALP for hydrolysis of PPi on the G4/NMM complex based sensing system for ALP (50 U/L) detection. Error bars were estimated from three replicate measurements.
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Fig. 4. Sensitivity of the sensing system for the detection of ALP. (A) Fluorescence spectra of the sensing system response to ALP with increasing concentrations, which are 0, 0.5, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 150 and 200 U/L, respectively. (B) Plot of fluorescence decrease intensities DF versus concentrations of ALP. DF ¼ F0 e F, F0 and F are the fluorescence intensities at 610 nm in the absence and presence of ALP, respectively. Error bars were estimated from three replicate measurements.
3.5. Selectivity for ALP detection Under the optimal conditions, the selectivity of the assay towards ALP was carried out in the presence of several interfering substances at a certain concentration, including Glu, GOx, PSA, BSA, IgG, pepsin and thrombin. Fig. 5 exhibits the fluorescence changes of the G4/NMM sensing system toward these interferents. One can observe that there are signicant differences between the large fluorescence change value generated by ALP and these tested interferents. These results demonstrate that the proposed assay is high selective for ALP detection towards other interferents. 3.6. ALP activity assay in human serum samples
human serum samples. Due to the complex composition of human serum, some containing proteins or amino acids may interfere with the detection of ALP. Fortunately, our method is highly sensitive and the interference can be reduced or avoided by diluting the serum. In this premise, different concentrations of ALP were spiked in 1% diluted human serum samples to evaluate the practical application of the proposed method. As shown in Table 1, the recovery efficiency in the range of 96.0e106.1% in different samples with RSD in the range of 3.5e5.9% can be obtained by the G4/NMM sensing system, indicating the effectiveness and reliability of the sensing system applied in real biological samples.
4. Conclusion
In order to testify the suitability and reliability of the sensing system for monitoring ALP activity in real biological samples, the analytical performance of this assay for ALP was conducted in
Fig. 5. Selectivity of the sensing system for the detection of ALP. Concentrations: ALP, 50 U/L; Glu, 10 mM; BSA and IgG, 0.2 mg/mL; GOx, PSA, Pepsin and Thrombin, 100 U/L. DF ¼ F0 - F, F0 and F are the fluorescence intensities at 610 nm in the absence and presence of ALP, respectively. Error bars were estimated from three replicate measurements.
In summary, a label-free and facile assay for sensitive and selective detection of ALP was proposed based on G-quadruplex with the assistance of Cu2þ. Cu2þ could quench the fluorescence of G4/ NMM complex effectively due to the proximal carboxylate groups of NMM incorporating to Cu2þ as well as the unfolding of Gquadruplex by Cu2þ. The higher affinity between PPi and Cu2þ could hinder the combining effect of Cu2þ with NMM, thus restoring the fluorescence. The presence of ALP catalyzed the hydrolysis of PPi to Pi and released the Cu2þ, leading to the fluorescence quenching once again. Based on the process of fluorescence “off-on-off” model of the G4/NMM comopex mediated by Cu2þ, a simple and cost-effective sensing platform for ALP was constructed which needs no complicated synthesis and modification procedure. The assay could quantitatively and selectively detect of ALP with a low detection limit of 0.3 U/L, which was further applied to detect of ALP in human serum samples successfully.
Declaration of competing interest There are no conflicts of interest to declare.
Acknowledgements Table 1 Recovery of the G4/NMM sensing system for ALP in human serum samples by standard addition method. Sample
Added (U/L)
Found (U/L)
Recovery (%)
RSD (n ¼ 3, %)
Serum I
1.0 10.0 50.0 1.0 10.0 50.0
1.04 10.32 50.64 0.96 10.61 52.15
104.0 103.2 101.3 96.0 106.1 104.3
4.6 4.9 5.8 5.9 4.6 3.5
Serum II
This work was supported by the National Natural Science Foundation of China (21775089) and the Natural Science Foundation of Shandong Province (ZR2017QB008, ZR2017JL010).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117607.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A label-free G-quadruplex-based fluorescence assay for sensitive detection of alkaline phosphatase with the assistance of Cu2þ Lin Ma, Xue Han, Lian Xia, Fengli Qu, Rong-Mei Kong* College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu Shandong, 273165, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2019 Received in revised form 7 August 2019 Accepted 6 October 2019 Available online 7 October 2019
The level of alkaline phosphate (ALP) is a significant biomarker index in organism. In this work, a labelfree and sensitive G-quadruplex fluorescence assay for monitoring ALP activity has been developed with the assistance of Cu2þ based on the competitive binding effect between pyrophosphate (PPi) and Gquadruplex-N-methylmesoporphyrin (G4/NMM) complex to Cu2þ. In the sensing assay, the G4/NMM complex is employed as a signal indicator, while the Cu2þ as a quencher and the PPi as recovery agent as well as the hydrolytic substance for ALP. In details, the fluorescence of the G4/NMM complex was efficiently quenched by introducing Cu2þ due to the proximal carboxylate groups of NMM coordinating with the Cu2þ as well as the unfolding of G-quadruplex by Cu2þ, while the higher affinity between PPi and Cu2þ could lead to the fluorescence recovery. However, in the presence of ALP, the PPi was hydrolyzed to phosphate ions (Pi) which cannot integrate with Cu2þ, resulting in the fluorescence quenching once again. Thus, a simple and facile way to inspect ALP has been exploited. The proposed assay shows a good linear relationship in the range from 0.5 to 100 U/L with the detection limit of 0.3 U/L. Moreover, the fabricated method is succeeded in detecting ALP in human serum samples, indicating the potential as a profitable candidate in biological and biomedical application. © 2019 Elsevier B.V. All rights reserved.
Keywords: G-quadruplex Label-free Alkaline phosphatase Cu2þ Fluorescence
1. Introduction Alkaline phosphate (ALP) is an essential metabolic enzyme with the function of catalyzing the dephosphorylation process [1,2]. As a widely distributed membrane-bound enzyme, ALP has broad substrate specificity that is able to hydrolyze a wide variety of phosphated compounds in vivo and in vitro. It is participated the dephosphorylation process of a variety of biomolecules, including nucleic acids, proteins and small molecules [3]. In another way, ALP is an indispensable biomarker in diseases’ diagnose. It is present widely in living tissues especially in liver, kidneys, prostate and bones. In the cycle of growth and apoptosis, ALP plays a key role in signal transduction and intracellular regulation [4]. The abnormal level of ALP in serum has been verified to be closely related to various diseases including diabetes, prostate cancer, bones disease and liver dysfunction [5e8]. Therefore, there is an urgent need that a sensitive and selective method should be developed to monitor ALP level. Up to now, a variety of facile strategies have been reported to determine of ALP level containing of colorimetry [9,10], surfaceenhanced Raman scattering [11], electrochemistry [12,13],
* Corresponding author. E-mail address: [email protected] (R.-M. Kong). https://doi.org/10.1016/j.saa.2019.117607 1386-1425/© 2019 Elsevier B.V. All rights reserved.
chemiluminescence [14] and fluorescence methods [15e18]. Compared to the traditional methods that have the defects of time-consuming and complicated operation, the fluorescence strategies for the detection of ALP have drawn wide attention because of the inherent merits such as higher sensitivity, easier devise and less modification. Many fluorescence assays have been done to explore high performance methods for the detection of ALP. For example, organic fluorophores [19,20], semiconductor quantum dots and metal nanoclusters [21e23] have been successfully applied to monitor ALP activity. However, most of these mentioned methods suffer from several limits such as low biocompatibility, costly reagents and complex synthesis routes. Therefore, develop simple, cost effective and more biocompatible fluorescent strategy for evaluating ALP level is still highly needed. Since ALP has broad substrate specificity, several substrates including different original or designed substrates have been employed as the hydrolyzed substances. There were adenosine triphosphate (ATP), pyrophosphate (PPi), p-nitrophenyl phosphate (PNPP), 4-methylumbellyferyl phosphate, 2-phosphate-L-ascorbic acid (AAP) and so on [24e26]. Therefore, by the catalyzing of the substrates hydrolysis, great progress has been made in developing sensitive and selective fluorescence methods for monitoring of ALP activity. Among many nature substrates for ALP, PPi has attracted much more attention due to its unique properties that it has a lower
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pH optimum compared to other substrates [27] and strongly chelate action with certain metal ions such as Cu2þ, Hg2þ and Zn2þ [28]. Traditional methods using radiolabeled PPi as ALP substrates were laborious with high detect limits rather than taking use of label-free substrate. Nowadays, various efforts were taking usage of label-free PPi to design ALP assays. For example, based on the inhibition of dsDNA-templated copper nanoparticles by PPi, Zhang et al. designed a label-free fluorescent strategy for the detection of ALP activity [23]. Liu et al. synthesized near-infrared fluoresence tryptophan-functionalized CuInS2 quantum dots (WeCuInS2 QDs) and used for the detection of ALP based on the quenching effect of ALP hydrolysis of PPi induced release of Cu2þ [29]. Although the mentioned fluorescence methods were effective for the detection of ALP, they are still suffering from the limits of complicated nanomertial synthesis procedure and the complex post-treatment process. Therefore, it is necessary to develop new simple and reliable methods for the determination of ALP with high sensitivity and selectivity. G-quadruplex is a structure which consists of stacking of planar G-tetrads [30]. Several small dye molecules combines with Gquadruplex can greatly enhanced the autologous fluorescence. Serving as the fluorescent signal output, G-quadruplex/dye molecule (G4/dye) complexes have been applied for the construction of multifarious biosensors with high biocompatibility. For example, zinc protoporphyrin IX (ZnPPIX), thioflavin T (ThT) and N-methylmesoporphyrin (NMM) are frequently employed as label-free G4/ dye molecule probes in detecting metal ions, DNA and small molecules [31e36]. Among these small dye molecules, NMM not lony is a commercially porphyrin derivative that has a pronounced structural selectivity for G-quadruplex with the increase of fluorescence rather than duplex, triplexes, double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), but also emissions fluorescence at the wavelength longer than 600 nm [37,38]. In such a case, when applied in biological system, the long wavelength fluorescence emission of the G4/NMM complex enables it to resist the interference of autofluorescence. In addition, the fluorescence of G4/NMM can be quenched by Cu2þ via the proximal carboxylate groups of NMM combining with Cu2þ as well as the unfolding of G-quadruplex by Cu2þ [39]. Inspired by the fact that the G4/NMM needs no more modification and can be simple manipulated, herein, a label-free fluorescence assay was established to monitor ALP level based on the quenching and recovery of the fluorescence of G4/NMM with the assistance of Cu2þ. The PPi was used for the inhibition of the quenching effect of Cu2þ to the fluorescence of G4/NMM due to the higher chelate affinity between PPi and Cu2þ. However, the hydrolyzation of PPi to phosphate ions (Pi) due to the presence of ALP resulted in the releasing of Cu2þ. The fluorescence of G4/NMM was requenched by the released free Cu2þ and the concentration of ALP was related to the fluorescence changes. Therefore, utilizing Gquadruplex without any modification, a type of label-free “off-onoff” fluorescence method was established for sensitive mornitoring of ALP activity. The assay was also applied for the detection of ALP in human serum samples with satisfactory results. Compared to the known nanomaterials based and molecular sensors for ALP reported in the literatures, our proposed approach possesses some remarkable features: (1) The DNA strands do not require any labeling, leading to less laborious and more cost-effective synthesis; (2) the commercially available NMM not only requires no complex synthesis but also emissions fluorescence at the wavelength longer than 600 nm when combined with G4. The red fluorescence emission feature enables it to resist the interference of autofluorescence when applied to biological sample analysis; and (3) the high sensitivity and specificity can be achieved via a simple “mix and detection” procedure, which are comparable or superior to the reported methods.
2. Experiments section 2.1. Reagents The oligonucleotide G4 (50 -GGGTTGGGCGGGATGGGG-30 , purified by HPLC) was synthesized and purchased from Takara Biotechnology Co. Ltd. (Dalian, China). N-methylmesoporphyrin (NMM, 96%), pyrophosphate (PPi, 99%) and alkaline phosphatase (ALP, lyophilized powder) were purchased from Beijing J&K Scientific Co., Ltd. (Beijing, China). The CuSO4$5H2O was purchased from Sinopharm Chemical Reagent Beijing Co. Ltd. Glucose (Glu), glucose oxidase (GOx), bovine serum albumin (BSA), pepsin, thrombin and immunoglobulin G (IgG) were purchased from Sigma-Aldrich. Prostatic specific antigen (PSA) was purchased from Shanghai Linc Bio Science Co. Ltd. Immunoglobulin G (IgG) was purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. The buffer used in this experiment was 25 mM HEPES buffer (pH 7.4), which was prepared by HEPES and HCl containing 100 mM NaCl, 10 mM KCl and 2.5 mM MgCl2$6H2O. All of the chemical reagents were analytical grade without any purification. The water used throughout all experiments was purified through a Millipore system (resistivity>18 MU cm). 2.2. Apparatus All the fluorescent spectrums were performed by F-7000 spectrometer (Hitachi, Japan). The experiment settings were as following: excitation wavelength was at 399 nm using 10/10 nm slit width, PMT detector voltage was 700 V. The emission spectra were recorded within the range of 580-700 nm. The pH measurement was carried out on a Mettler-Toledo Delta 320 pH meter at room temperature (about 25 C). 2.3. Preparation of G4/NMM Before the experiment, the G4 oligonucleotide was heated at 95 C for 5 min and then cooled down at 4 C in the refrigerator. G4/ NMM was achieved by diluting the stock solution of G4 and NMM with 25 mM HEPES buffer. The mixture was incubated at 37 C for 30 min to allow the formation of G4/NMM. 2.4. Fluorescence detection of PPi To verify the inhibition of the PPi to the quenching effect of Cu2þ, the fluoresence of G4/NMM was investigated in the presence of different concentrations of PPi. First, 10 mL of 100 mM Cu2þ was premixed with 10 mL of PPi with different concentrations. Then, the Cu2þ-PPi complexs were introduced to the above prepared G4/ NMM solutions and incubated for 25 min at room temperature. Finally, the fluorescence spectra of these solutions were recorded in the wavelength rang of 580-700 nm. 2.5. Fluorescence detection of ALP Fluorescence detection of ALP was conducted in 500 mL Eppendorf tubes. 5 mL of ALP with various concentrations and 5 mL of PPi (1.2 mM) were premixed at 37 C for 30 min and then 10 mL of Cu2þ (100 mM) was added into the mixture at room temperature. Finally, the mixture was added to the solution that containing 20 mL of G4/ NMM and 160 mL HEPES buffer, and incubated at room temperature for 25 min. The fluorescence spectra of the mixture were recorded in the wavelength rang of 580-700 nm. The real complex samples for detecting ALP were conducted in diluted human serum samples, and the detection procedure was similar to the above experiments in buffer solution just containing 1% diluted human serum including 5 mM Cu2þ and 30 mM PPi. Serum sample experiments
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were performed according to the Guidelines for Ethical Committee, Qufu Normal University. All studies were approved by Ethical Committee, Qufu Normal University. Informed consents were obtained from human participants of this study. 3. Results and discussion 3.1. Mechanism of the assay for ALP detection The principle of the designed strategy to detect ALP based on label-free G-quadruplex and competing binding reaction with the mediate of the Cu2þ was depicted in Scheme 1. As is known to all, the fluorescence of some dye molecules such as NMM can be extremely enhanced by combining with G-quadruplex to form complex [37]. In addition, the fluorescence of G4/NMM can be quenched by Cu2þ via the proximal carboxylate groups of NMM combining with Cu2þ as well as the unfolding of G4 by Cu2þ, with the association constant (Ka) of NMM with Cu2þ is 1.82 106 M-1 [39]. Inspired of the point, a label-free assay was developed for the detection of ALP based on the inhibition of PPi to the quenching effect of Cu2þ to G4/NMM complex. In details, the G4/NMM complex fluorescence could be quenched due to the proximal carboxylate groups coordinating with the Cu2þ to form Cu2þ/NMM. In another way, Cu2þ caused the unfolding of G-quadruplex as well as induced the fluorescence quenching of G4/NMM complex. The higher association between PPi and Cu2þ (1.0 109 M-1) disturbed the interaction between Cu2þ and G4/NMM, thus leading to the fluorescence recovering. The introduction of the ALP would hydrolyze PPi into Pi so that the Cu2þ was released into solution. The free Cu2þ combined with G4/NMM complex, which resulted in the fluorescence quenching again. As a whole, in the presence of Cu2þ and PPi, the fluorescence was recovered, while the introduction of ALP induced the fluorescence quenching reoccurs. Since the fluorescence quenching of the G4/NMM complex was related to the concentration of ALP, the proposed principle is effective to monitor ALP level. Meanwhile, no complicated synthesis procedure and modification make the proposed label-free assay simple and costeffective. 3.2. Feasibility of the designed strategy As a proof of concept to evaluate the feasibility of our design for ALP detection, the fluorescence experiments were carried out
Scheme 1. Schematic illustration of the label-free detection strategy for ALP based on the G4/NMM complex with the assistance of Cu2þ and PPi.
3
under different conditions. As is shown in the Fig. 1, the G4/NMM complex exhibited strong fluorescence at 610 nm (curve a). A significant quenching of the G4/NMM fluorescence can be observed when the Cu2þ was added into the solution (curve b). Furthermore, after the addition of PPi, the fluorescence was obviously recovered (curve c). The results show that the G4/NMM complex exhibited an obvious “off-on” phenomenon with the assistance of Cu2þ and PPi. However, with a certain amount of ALP was introduced into the mixture including G4/NMM, Cu2þ and PPi, one can obvious observe the fluorescence “on-off” phenomenon (curve d) due to hydrolyzation of PPi to Pi by ALP induced releasing of free Cu2þ. Therefore, the results indicate that the proposed label-free can be used for the effective detection of ALP.
3.3. Optimization of detection conditions To achieve the best analytical performance of the proposed assay for ALP detection, some detection parameters involved in this assay were optimized. Since the fluorescence enhancement was directly related to the increasing concentration of G-quadruplex, the fluorescence experiment was first carried out in the presence of NMM at a fixed concentration of 0.3 mM. As shown in Fig. 2, with the increase of G4 concentrations range from 0 to 0.4 mM, the fluorescence intensity increases gradually and can reach the plateau at the G4 concentration of 0.3 mM, indicating the complete binding of NMM by G4. It is obvious to know that the redundant G4 cannot cause the further fluorescence increasing, which may be due to the binding concentration ratio between NMM and Gquadruplex is 1:1 [39]. For the phenomenon, 0.3 mM G4 was chosen for the further experiments. Then, the related experiment conditions of the G4/NMM fluorescent sensing platform for the detection of ALP activity with the assistance of Cu2þ were optimized. Quantative detection of ALP was based the “off-on-off” model of the G4/NMM fluorescence. Therefore, the optimal concentration of Cu2þ used to quench the fluorescence of G4/NMM was investigated. As shown in Fig. 3A, with the increase of Cu2þ concentration, the fluorescence of G4/NMM at 610 nm was decreased gradually due to the coordination of NMM with Cu2þ as well as the unfolding of G-quadruplex by Cu2þ. The quenching efficiency could even achieve as high as 89% under the concentration of 3 mM Cu2þ. The time dependence of the fluorescence after the addition of Cu2þ was studied due to that the fluorescence quenching was significantly related with the binding time between Cu2þ and NMM. The Fig. 3B shows that the fluorescence quenching reaches the plateau when the incubation time reaches
Fig. 1. Fluorescence spectra under different conditions: (a) G4/NMM, (b) G4/ NMM þ Cu2þ, (c) G4/NMM þ Cu2þ þ PPi, (d) G4/NMM þ Cu2þ þ PPi þ ALP. The final concentrations of G4/NMM, Cu2þ, PPi and ALP are 0.1 mM, 5 mM, 30 mM and 100 U/L, respectively.
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enzymatic reaction time of ALP for the hydrolysis of PPi was also very important for the G4/NMM complex based sensing system. According to the experiment results shown in Fig. 3D, the catalytic hydrolysis of PPi can be completed within 30 min.
3.4. Analytical performance of sensing strategy for ALP detection
Fig. 2. Fluorescence intensities of G4/NMM at 610 nm. The final concentration of NMM is 0.3 mM. Error bars were estimated from three replicate measurements.
25 min, implying the appropriate time of Cu2þ quenching was 25 min in the designed system. Thus, 3 mM Cu2þ and 25 min incubation time of Cu2þ are choosen for the following experiments. Next, the optimized Cu2þ concentration was used to investigate the fluorescence changes of the G4/NMM complex by adding Cu2þPPi complex with the PPi concentrations range from 0 to 50 mM. As shown in Fig. 3C, the fluorescence intensity gradually increases with the addition of PPi from 0 to 30 mM, and then kept almost unchanged, indicating that the fluorescence recovery proceeded in a dose dependent response to PPi. The luorescence recovery could be reached a zenith at PPi concentration of 30 mM, which was employed in the subsequent experiments. Additionally, the
To testify the ability of the Cu2þ-mediated G4/NMM assay for ALP detection, various different concentration of ALP under optimized conditions was investigated. Fig. 4A depicts the fluorescence spectra of G4/NMM sensing system for the detection of ALP with the concentrations rang from 0 to 200 U/L. It is clearly observed that the peak intensity decreased gradually with increasing of ALP concentration. As shown in Fig. 4B, the plot of the peak intensity of G4/NMM complex shows an excellent linear correlation versus the ALP concentrations range from 0.5 to 100 U/L. The calibration equation was DF ¼ 3.08 þ 7.03[ALP] (R2 ¼ 0.995), where DF is the fluorescence decrease intensity of G4/NMM at 610 nm between the absence and presence of ALP with different concentrations, [ALP] is the concentration of ALP. A detection limit of 0.3 U/L can be obtained from the calibration curve according to the definition of detection limit (3s/slope). The sensitivity is enough for ALP detection in biological samples as the normal range of serum ALP in adults is about 46-190 U/L [40]. In addition, the analytical performance of proposed assay was compared to the previously reported for ALP in Table S1. The proposed biosensor revealed superior characteristic in wide linear range and low detection limit. The results indicated the proposed label-free G4/NMM complex based sengsing system has the potential to be extended to practical fluorescence detection of ALP.
Fig. 3. (A) The effect of the amount of Cu2þ on the fluorescence intensity of G4/NMM complex. (B) The effect of the incubation time of Cu2þ with G4/NMM on the fluorescence intensity of G4/NMM complex. (C) The effect of the amount of Cu2þ-PPi complex with a fixed Cu2þ concentration at 3 mM and different amounts of PPi. (D) The effect of the enzymatic reaction time of ALP for hydrolysis of PPi on the G4/NMM complex based sensing system for ALP (50 U/L) detection. Error bars were estimated from three replicate measurements.
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Fig. 4. Sensitivity of the sensing system for the detection of ALP. (A) Fluorescence spectra of the sensing system response to ALP with increasing concentrations, which are 0, 0.5, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 150 and 200 U/L, respectively. (B) Plot of fluorescence decrease intensities DF versus concentrations of ALP. DF ¼ F0 e F, F0 and F are the fluorescence intensities at 610 nm in the absence and presence of ALP, respectively. Error bars were estimated from three replicate measurements.
3.5. Selectivity for ALP detection Under the optimal conditions, the selectivity of the assay towards ALP was carried out in the presence of several interfering substances at a certain concentration, including Glu, GOx, PSA, BSA, IgG, pepsin and thrombin. Fig. 5 exhibits the fluorescence changes of the G4/NMM sensing system toward these interferents. One can observe that there are signicant differences between the large fluorescence change value generated by ALP and these tested interferents. These results demonstrate that the proposed assay is high selective for ALP detection towards other interferents. 3.6. ALP activity assay in human serum samples
human serum samples. Due to the complex composition of human serum, some containing proteins or amino acids may interfere with the detection of ALP. Fortunately, our method is highly sensitive and the interference can be reduced or avoided by diluting the serum. In this premise, different concentrations of ALP were spiked in 1% diluted human serum samples to evaluate the practical application of the proposed method. As shown in Table 1, the recovery efficiency in the range of 96.0e106.1% in different samples with RSD in the range of 3.5e5.9% can be obtained by the G4/NMM sensing system, indicating the effectiveness and reliability of the sensing system applied in real biological samples.
4. Conclusion
In order to testify the suitability and reliability of the sensing system for monitoring ALP activity in real biological samples, the analytical performance of this assay for ALP was conducted in
Fig. 5. Selectivity of the sensing system for the detection of ALP. Concentrations: ALP, 50 U/L; Glu, 10 mM; BSA and IgG, 0.2 mg/mL; GOx, PSA, Pepsin and Thrombin, 100 U/L. DF ¼ F0 - F, F0 and F are the fluorescence intensities at 610 nm in the absence and presence of ALP, respectively. Error bars were estimated from three replicate measurements.
In summary, a label-free and facile assay for sensitive and selective detection of ALP was proposed based on G-quadruplex with the assistance of Cu2þ. Cu2þ could quench the fluorescence of G4/ NMM complex effectively due to the proximal carboxylate groups of NMM incorporating to Cu2þ as well as the unfolding of Gquadruplex by Cu2þ. The higher affinity between PPi and Cu2þ could hinder the combining effect of Cu2þ with NMM, thus restoring the fluorescence. The presence of ALP catalyzed the hydrolysis of PPi to Pi and released the Cu2þ, leading to the fluorescence quenching once again. Based on the process of fluorescence “off-on-off” model of the G4/NMM comopex mediated by Cu2þ, a simple and cost-effective sensing platform for ALP was constructed which needs no complicated synthesis and modification procedure. The assay could quantitatively and selectively detect of ALP with a low detection limit of 0.3 U/L, which was further applied to detect of ALP in human serum samples successfully.
Declaration of competing interest There are no conflicts of interest to declare.
Acknowledgements Table 1 Recovery of the G4/NMM sensing system for ALP in human serum samples by standard addition method. Sample
Added (U/L)
Found (U/L)
Recovery (%)
RSD (n ¼ 3, %)
Serum I
1.0 10.0 50.0 1.0 10.0 50.0
1.04 10.32 50.64 0.96 10.61 52.15
104.0 103.2 101.3 96.0 106.1 104.3
4.6 4.9 5.8 5.9 4.6 3.5
Serum II
This work was supported by the National Natural Science Foundation of China (21775089) and the Natural Science Foundation of Shandong Province (ZR2017QB008, ZR2017JL010).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117607.
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L. Ma et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 227 (2020) 117607
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