- Email: [email protected]
GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay
Accepted Manuscript Title: GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay Authors: Ying Shi, Miao Yang, Li Liu, Yanjiao Pang, Yijuan Long, Huzhi Zheng PII: DOI: Reference:
S0925-4005(18)31469-2 https://doi.org/10.1016/j.snb.2018.08.038 SNB 25185
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
27-3-2018 3-8-2018 7-8-2018
Please cite this article as: Shi Y, Yang M, Liu L, Pang Y, Long Y, Zheng H, GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.08.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay
IP T
Ying Shi1, Miao Yang1, Li Liu1,2, Yanjiao Pang1, Yijuan Long1, Huzhi Zheng1,* 1
SC R
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest
University), Ministry of Education, College of Chemistry and Chemical Engineering
U
Southwest University Beibei, Chongqing, 400715, P. R. China.
2
N
College of Chemistry and Environmental Science Qujing Normal University Qujing,
A
655011, P. R. China.
M
*Corresponding author: Huzhi Zheng, Email: [email protected]
PT
ED
Graphical Abstract
We showed for the first time that GTP exhibited peroxidase-like activity and a
CC E
colorimetric platform for ALP and AFP detection was designed based on the enzymatic cascade reaction by coupling ALP-catalyzed dephosphorylation of GTP
A
with GTP-catalyzed oxidation of TMB.
IP T SC R U
Highlights
GTP was first found to exhibit an intrinsic peroxidase-like activity.
The peroxidase-like activity of GTP was closely related to the phosphate group.
A sensitive colorimetric platform was designed for ALP and AFP sensing based on enzymatic cascade reaction.
This finding provides further insights into new biological function of GTP and construction of biosensors based on the enzyme-like activity of GTP.
A
CC E
PT
ED
M
A
N
Abstract: Guanosine triphosphate (GTP), a substrate for RNA synthesis, plays a pivotal role in life-form metabolism at the cellular level. Herein, we first showed that GTP exhibited an intrinsic peroxidase-mimic activity, accelerating H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a color reaction. Compared to GTP, weaker catalytic activity was observed from guanosine diphosphate (GDP) and virtually no catalytic activity was observed from guanosine monophosphate
IP T
(GMP) and guanosine. Alkaline phosphatase (ALP) can catalyze the dephosphorylation
of GTP with in situ generation of GDP, GMP and guanosine, which followed by
SC R
receding the oxidation of TMB. Inspired by this, a novel colorimetric sensor for ALP
detection was designed based on the enzymatic cascade reaction by coupling ALPcatalyzed dephosphorylation of GTP with GTP-catalyzed oxidation of TMB. This
U
method allowed ALP sensing in a range from 0.01 to 100 U/L with a detection limit of
N
0.009 U/L, which was sensitive enough for ALP activity assay in biological samples
A
(the normal ALP level range of adult serum is 20-140 U/L). Moreover, ALP is widely used in enzyme-linked immunosorbent assays (ELISA) as a signal reporter, the proof-
M
of-concept ELISA for alpha-fetoprotein (AFP) detection was established. This
ED
unexpected discovery not only extends the new biological function of GTP, but also holds great promise for development of versatile biosensors that initiate the
PT
concentration changes of GTP.
A
CC E
Keywords: Guanosine triphosphate; Peroxidase-like activity; Alkaline phosphatase; Alpha-fetoprotein; ELISA
1. Introduction Guanosine triphosphate (GTP), well-known as a member of fundamental building blocks for synthesis of RNA during the transcription process, plays a key role in the regulation of a large array of cellular process and biochemical pathway.[1, 2] For example, GTP can act as a source of energy like adenosine triphosphate (ATP) in the course of biological metabolism, but it more specific for protein synthesis.[3] GTP
IP T
also has the role in signal transduction with G-proteins, during which GTP is
converted to guanosine diphosphate (GDP) through the action of GTPases that
SC R
relating to a variety of human diseases such as cancer.[4-7] In addition, GTP is
involved in citric acid cycle referred to energy transfer in the cell metabolism.[8, 9] However, despite its orthodox role in life activities, experimental attempts to observe
U
the new biological function of GTP remain sparse.
N
Alkaline phosphatase (ALP, EC 3.1.3.1), widespread in liver, bone and kidney, is a non-specific hydrolase that catalyses the dephosphorylation process of broad substrates
A
including proteins, nucleic acids and small molecules that contain phosphate functional
M
groups in alkali condition.[10, 11] It plays a significant role in regulating phosphate metabolism involved in cell cycle, growth, apoptosis and signal transduction
ED
pathways in biological system.[12] The abnormal expression level of ALP in serum can be identified as a diagnostic indicator for a series of diseases such as bone diseases
PT
(osteoblastic bone cancer, Paget’s disease, ),[13, 14] liver dysfunction (cancer, hepatitis, and obstructive jaundice),[15, 16] breast and prostatic cancer,[17] and diabetes[18] etc.
CC E
Moreover, despite the physiological functions of ALP in related biological processes, it is also commonly used as an enzyme-label within enzyme-linked immunosorbent assays (ELISA) for protein biomarker detection owing to being easily conjugated to
A
antibodies, inexpensive, broad substrate specificity and commercial availability and high catalytic activity.[12, 19, 20] Here, in the present work we first demonstrated that GTP exhibited the catalytical activity like nature horseradish peroxidase (HRP), catalysing the oxidation of the peroxidase substrates 3,3′,5,5′-tetramethylbenzidine (TMB) to produce colour reaction in the presence of H2O2. Following a series of experiments, we sought to investigate
and prove this intriguingly discovery. The peroxidase-like activity of GTP is indeed attributed to GTP itself as a catalyst rather than a reactant during the reaction. Meanwhile, experiments were also extended to GTP analogue, i.e., guanosine diphosphate (GDP), guanosine monophosphate (GMP) and guanosine. The results demonstrated that the peroxidase-like activity of GTP and its analogue were depended on the phosphate group and the catalytic ability rapidly weakened or even lost along
IP T
with the decrease of phosphate group: weaker catalytic activity was observed from guanosine diphosphate (GDP) and virtually no catalytic activity was observed from
SC R
guanosine monophosphate (GMP) and guanosine under the same condition.
Inspired by this significant finding, a colorimetric platform for ALP detection was designed based on GTP-mediated enzymatic cascade reaction. Upon addition of ALP,
U
GTP can be hydrolyzed to generate the product GDP with weaker catalytic activity or
N
GMP and guanosine with no catalytic activity, and thereby leading to the declined readout signal of oxidized TMB (oxTMB). Then, as a proof of concept, ALP-labelled
A
enzyme-linked immunosorbent assay for alpha fetoprotein (AFP) was designed
M
coupling with the biocatalytic activity of GTP. The proposed immunoassay has been applied for AFP level detection in serum samples with satisfied results. This significant
ED
discovery not only extends the new biological function of GTP, but also opens new opportunities to impel the development of biosensors with triggering the variation of
PT
GTP concentration.
CC E
2. Material and methods 2.1. Chemicals
Guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine
A
monophosphate (GMP), guanosine (99%) and 3,3′,5,5′-tetramethylbenzidine (TMB) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Alkaline phosphtase (ALP) was purchased from Sigma-Aldrich (St. Louis, MO). Human AFP antigen and antibody were purchased from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Hydrogen peroxide (H2O2) was obtained from Chongqing Chuandong
Chemical Co., Ltd. (Chongqing, China). All the commercial available regents were used without further purification. All solutions were prepared using ultrapure water (18.2 MΩ·cm-1) from a Milli-Q automatic ultrapure water system. 2.2. Peroxidase activity assays GTP to functionally mimic peroxidase was evaluated by the catalytic oxidation of
IP T
organic substrate TMB. Briefly, 100 μL of 100 μM GTP, 100 μL of 8.0 mM organic
peroxidase substrates and 100 μL of 100 mM H2O2 were added into pH 5.0 phosphate
SC R
buffer solution (PBS, 20 mM) to a volume of 1.0 mL to produce color product. After incubation under the optimal condition, the absorption spectra of the solution were recorded on a UV-2450 spectrometer (Shimadzu, Japan).
U
2.3. Electron spin resonance spectroscopy
N
Electron spin resonance spectroscopy (ESR) was used to detect hydroxyl radical
A
(•OH) generation with spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The
M
reaction mixture was composed of 50 mM DMPO, 10 mM H2O2, and different concentration of GTP in pH 5.0 PBS. Data collection began after sample mixing for 1
ED
min and the ESR spectra were recorded on a Bruker ESR 300E using the following settings: 20 mW microwave power, 1 G field modulation, and 100 G scan range.
PT
2.4. GTP hydrolysis and free inorganic phosphate determination GTP hydrolysis degree was measured using a malachite green-ammonium
CC E
molybdate assay.[21] A typical procedure was carried out as follows: (1) 500 μL of GTP (10 μM) was incubated at different temperatures for 40 min with the solution at 4°C as a blank. (2) Malachite green (0.081%), polyvinyl alcohol (2.3%), ammonium
A
heptamolybdate tetrahydrate (5.7% in 6 M HCl) and water were mixed in a ratio of 2:1:1:2. (3) Subsequently, 100 μL of the above obtained mixture solution was dropped into the GTP solution with adding 40 μL of 3.4% sodium citrate to halt the hydrolysis of GTP. (4) Then the reaction was allowed to proceed for 15 min and the absorbance values at 620 nm were recorded. In addition, a standard curve for phosphate was
obtained using different NaH2PO4 concentrations, and the amount of inorganic phosphate generated by GTP hydrolysis was calculated using the standard curve. 2.5. Ultra-performance liquid chromatography-mass spectrum (UPLC-MS) Analysis Ultra-performance liquid chromatography (UPLC) was carried out on an Acquity
IP T
UPLC system (Waters, UK). Separation was performed on an Acquity BEH C18
column (2.1 × 50 mm, 1.7 μm particle size) with column oven temperature at 40°C.
SC R
The mobile phases were: 80% acetonitrile and 20% methanol. The flow rate was 0.2 mL/min. Separated substances were detected by Xevo TQ-S mass spectrometer
(Waters, UK) with an electrospray ionization source. Source conditions were fixed as
U
follows: source temperature, 150°C; desolvation temperature, 350°C; cone voltage, 35
N
eV; capillary voltage, 2.5 kV.
A
2.6. Kinetic analysis
M
Steady-state kinetic assays for the catalytic oxidation of TMB by GTP were carried out by monitoring the absorbance change at 652 nm at selected time intervals.
ED
Experiments were carried out using 10 μM GTP at the optimal temperature in a reaction volume of 4.0 mL pH 5.0 PBS by varying concentrations of TMB at a fixed
PT
concentration of H2O2 or vice versa. The kinetic parameters were calculated based on Michaelis-Menten equation: v = Vmax•[S] /([S]+ Km), where v is the initial velocity,
CC E
Vmax is the maximal reaction velocity, and [S] is the concentration of substrate and Km is the Michaelis constant.
A
2.7. ALP detection The ALP activity assay was conducted based on its enzymatic hydrolysis of GTP to
generate GDP, GMP and guanosine. First, 10 μL of ALP with different activities ranging from 0 to 200 U/L, 10 μL of 100 μM GTP was added into 30 μL of 10 mM pH 8.0 Tris-HCl buffer (containing 5 mM MgCl2 and 0.1 mM ZnCl2) for an incubation time of 1 h at 40°C. After that, 100 μL of 8.0 mM TMB, 100 μL of 100
mM H2O2, and 100 μL of pH 5.0 PBS were added in the above solution and diluted to 1.0 mL. Finally, the mixture was incubated at 50°C for 40 min and the absorbance of the oxTMB at 652 nm was recorded. 2.8. Immunoassay for AFP First, 100 µL of 0.50 μg/mL capture antibody (Ab1) dissolved in 0.050 M NaHCO3-
IP T
Na2CO3 buffer (pH 9.6) was added to 96-well microplates and incubated at 4°C
overnight followed by rinsing with the washing buffer (0.010 M PBS containing
SC R
0.05% Tween 20, pH 7.4) for five times. Then the wells were blocked with 100 μL
1% bovine serum albumin (BSA) at 37°C for 1 h and washed with the washing buffer thoroughly. Following that, 100 μL of AFP with different concentrations were added
U
into the wells and incubated at 37°C for 90 min followed by washing. Subsequently, 100 μL of 2.0 μg/mL biotinylated-antibody (Ab2) were added into each well for an
N
incubation time of 30 min at 37°C and then washed five times with washing buffer.
A
Then 100 μL of avidin-ALP (1:500) were added to conjugate to antibody through
M
biotin-avidin reaction for an incubation time of 30 min at 37°C and again washed thoroughly. After the formation of sandwich structure, 20 μL of 15 μM GTP in 10
ED
mM Tris-HCl buffer (pH 8.0) containing 5 mM MgCl2 and 0.1 mM ZnCl2 was added and incubated at 40°C for 60 min. Finally, 30 μL of 8.0 mM TMB, 30 μL of 100 mM
PT
H2O2, 30 μL of pH 3.0 PBS buffer, and 190 μL of ultrapure water were added into the 96 well plates and reacted at 50°C for 40 min before the absorbance of oxTMB at 652
CC E
nm was measured.
3. Results and discussion
A
3.1. Peroxidase-like properties of GTP GTP could apply as a catalyst for the oxidation of peroxidase substrates TMB to
oxidized TMB (oxTMB) in the presence of H2O2 (Fig. 1a). As shown in Fig. 1b, a characteristic absorbance of oxTMB at 652 nm can be observed upon adding GTP into TMB-H2O2 system, while there was negligible absorbance variation in the absence of GTP, TMB, or H2O2. Fig. 1c displayed that the increasing concentration of GTP could
improve the reaction rates, reaching a plateau at about 10 μM. These results demonstrated that GTP can function as peroxidase mimics. Meanwhile, we found that the catalytic efficiency of GTP, like HRP, dependent on pH, temperature, H2O2 concentrations and incubation time.[22] As shown in Fig. S1, the optimal pH, temperature, H2O2 concentration and reaction time for catalytic activity of GTP were evaluated to be 5.0, 50°C, 10 mM and 40 min, respectively. Note that GTP
IP T
required a high H2O2 concentration to reach the maximum level of peroxidase-like activity and then remain stable with increased H2O2 concentration while natural enzyme
SC R
HRP was denatured at high H2O2 concentration, suggesting that the catalytic activity of GTP exhibited higher tolerance level at high H2O2 concentration than HRP. [22, 23] Fig. 1
U
3.2. Activity is due to intact GTP not hidrolysis releasing energy
N
Qu et al. reported that GTP can improve the oxidase-like activity of nanoceria due
A
to the hydrolysis of GTP coupling with releasing energy.[24] It is important to
M
exclude that the hydrolysis of GTP render its ability to promote the TMB-H2O2 reaction. Then hydrolysis degree was evaluated by measuring the amount of inorganic
ED
phosphate released using a malachite green-ammonium molybdate assay.[21] As shown in Fig. S2a, the hydrolysis amounts of GTP taken place under our experimental
PT
temperature were no more than 0.55 μM, that is, hydrolysis degree could not exceed 5.5%, suggesting that little energy would release and this change in the reaction was
CC E
insignificant.
UPLC-MS was conducted to further ascertain the integrity of GTP during the
reaction process, i. e., GTP can act as catalyst to accelerate the TMB-H2O2 reaction.
A
As shown in Fig. S2b, the curve 1, a single peak (fragment ion at m/z 158.86) with retention time at 0.92 min, display the UPLC-MS analysis of GTP in the absence of TMB and H2O2 as a control. Curve 2 illustrates the separation result of GTP mediated TMB-H2O2 reaction. For the two peaks observed, one with the same retention time (0.91 min) corresponds to GTP and another peak (fragment ion at m/z 195.11) with
retention time at 3.8 min corresponds to oxTMB. In addition, the peak area of GTP in curve 2 (32555) was almost identical to that in curve 1 (31345). The sum of evidences pointed to that GTP changed little in either quality or quantity during the reaction and supported that GTP indeed act as a catalyst to mimic peroxidase. 3.3. Production of hydroxyl radicals catalyzed by GTP
IP T
Formation of reactive oxygen species (ROS) is of vital importance in catalytic oxidation chemistry. To identify the kind of ROS formation catalyzed by GTP,
SC R
different radical scavengers including ascorbic acid (AA), sodium azide (NaN3) and superoxide dismutase (SOD) were introduced to GTP-TMB-H2O2 system. As
illustrated in Fig. 2a, little variation in the absorbance of oxTMB at 652 nm was caused with addition of varied concentration of NaN3 and SOD, which were used as
U
scavengers for 1O2 and O2•−, respectively.[25,26] Whereas, the absorbance values
N
decreased progressively with the addition of increased concentration of AA, an
A
effective •OH and O2•− scavengers.[27] These results demonstrated that •OH was
M
generated during the GTP mediated-catalytic reaction.
ED
Electron spin resonance (ESR) is a powerful and direct technique for ROS qualitative analysis using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. In the presence of •OH, DMPO can capture the short-lived •OH to form the DMPO/•OH
PT
spin adduct, which is a relatively stable paramagnetic product with a characteristic ESR signal of a 1:2:2:1 quartet.[28] As shown in Fig. 2b, H2O2 added alone showed
CC E
poor DMPO/•OH adduct signal intensity, but the characteristic signal was enhanced by adding GTP and found to be progressively increased with the increasing concentration, suggesting that GTP possessed the peroxidase-mimic ability to
A
decompose H2O2 to generate reactive •OH radicals. Fig. 2
3.4. Kinetics assay The peroxidase-like catalytic mechanism and kinetic parameters of GTP were further investigated using steady-state kinetics. As shown in Fig. 3, the oxidation
reaction process catalyzed by GTP could be modeled phenomenologically following the conventional enzymatic dynamic regulation of the Michiaelis-Menten equation v = Vmax•[S] /([S]+ Km), where v is the initial velocity, Vmax is the maximal reaction velocity, Km is the Michaelis constant and [S] is the concentration of the substrate. We also compared the kinetic parameters of GTP with other reported small molecular enzyme-mimic: fluorescein (F)[29] and its derivatives including 5(6)-
IP T
carboxyfluorescein (CF), 5(6)-aminofluorescein (AF)[30], 2', 7'-difluorofluorescein (DFF) [31] and 2′,7′- dichlorofluorescein (DCF)[32] as well as acidic amino acids
SC R
including L-glutamic acid (L-Glu) and L-aspartic acid (L-Asp)[33]. As summarized in Table 1, the apparent Km value, indicating the binding affinity between enzyme and substrates, of GTP toward H2O2 was lower than that of F and its derivatives except for
U
DCF and acidic amino acids, suggesting that GTP had a better affinity to H2O2. The
N
Vmax value of GTP was higher and the catalytic constant kcat of GTP was two orders of
A
magnitude higher that of fluorescein and its derivatives and four orders of magnitude higher than that of acidic amino acid. From these observations it is evident that the
M
catalytic activity of GTP is more efficient than other small molecular enzyme-mimics.
ED
However, the kcat value of GTP is six orders of magnitude lower than that of natural enzyme HRP. Thus, it is still a great challenge to design and screen small molecular
Fig. 3 Table 1
CC E
PT
mimetic enzymes with higher catalytic activity.
3.5. ALP sensing based on peroxidase-like GTP ALP, an essential enzyme in phosphate metabolism, can catalyze the hydrolysis of
A
phosphoryl eaters and the abnormal expression level was associated with several diseases.[12] GTP as one of the typical substrates of ALP can be hydrolyzed to yield GDP, GMP and guanosine phosphate[2] and we envisioned that ALP can be detected based on the peroxidase-like activity of GTP. To verify the feasibility of this assay, we evaluated that whether GDP, GMP and guanosine could catalyze the oxidation of TMB in the presence of H2O2 under the same experiment condition. As shown in Fig.
S3, only GDP possessed the peroxidase-like activity but one time lower than GTP, i.e., the catalytic ability rapidly weakened or even lost along with the decrease of phosphate group. These observations fully supported that ALP activity sensing platform can be accordingly constructed based on the significance level of difference between GTP and its hydrolysis product.
IP T
The response progress of GTP-mediated enzymatic cascade reaction for ALP detection was illustrated in Fig. 4a, upon addition of ALP, GTP with high catalytic activity was hydrolyzed to generate GDP with weaker catalytic activity and GMP
SC R
guanosine non-catalytic activity to further regulate the readout signal of oxidized
TMB. The absorption spectra were recorded by varying ALP concentration from 0.01 to 200 U/L and the absorbance decreased gradually upon enhancing ALP level as a
U
result of the consumption of GTP (Fig. 4b). The relationship between the absorbance
N
changes of this colorimetric sensor and the ALP activity was depicted in Fig. 4c, the
A
absorbance changes was plotted as a function of ALP level in a range from 0.01-100
M
U/L (Fig. 4d) with a detection limit of 0.009 U/L, which was sensitive enough for ALP activity assay in biological samples (the normal ALP level range of adult serum
ED
is 20-140 U/L).[34]
Then the selectivity of this GTP-based ALP sensing platform was evaluated by
PT
separated addition of several proteins including bovine serum albumin (BSA), lysozyme, glucose oxidase (GOx), uricase, immunoglobulin G (IgG), trypsin and
CC E
esterase under the same condition. The concentration was 200 U/L for ALP and 0.10 mg/mL for each other interfering proteins. As shown in Fig. 4e, all the interference proteins showed no hydrolysis on GTP and the peroxidase activity of GTP was
A
maintained, demonstrating the high selectivity of the proposed sensing approach. Fig. 4
Given the importance of screening enzyme regulators in clinical chemistry and drug development, Na3VO4, a common ALP inhibitor was selected for the inhibiting assay.[12] As shown in Figure S4, the ALP activity decreased with the addition of increased Na3VO4 concentration and the IC50 value (half-maximal inhibitory
concentration) for 500 U/L ALP was determined to be 35 μM, demonstrating the feasibility of this protocol for screening ALP inhibitor in drug discovery. 3.6. ALP-linked immunoassay for AFP Considering the significance of ALP as the most commonly enzyme label in enzyme-linked immunosorbent assays due to its high catalytic activity, broad
IP T
substrate specificity and commercial availability,[12] ALP-based immunoassay
integrating with the peroxidase-like activity of GTP was established. In this study,
SC R
AFP, a well-known biomarker of some cancerous diseases in clinical diagnosis,[3537] was used as a model analyte in our proof-of-concept experiment. As
schematically illustrated in Fig. 5a, capture antibody (Ab1) was immobilized on the
U
microtiter plates to recognize target antigen AFP, then biotinylated-antibody (Ab2) was further conjugated and a sandwich structure can be formed. Subsequently, ALP
N
can be anchored on the sandwich structure through the specific reaction between
A
biotin and avidin and further causing the depletion of GTP due to hydrolysis of
M
labeled ALP, which eventually leading to weaken the oxidation of TMB. As shown in Fig. 5c, the absorbance gradually decreased with the target antigen AFP concentration
ED
rising and a linear relationship can be obtained in the range from 1.0-100 ng/mL with a detection limit of 0.5 ng/mL (Fig. 5d). The color responses to different
PT
concentrations of AFP can be seen from the photos in Fig. 5b, an obvious color changed can be observed with naked eye compared to the blank color when the
CC E
concentration of AFP was 20 ng/mL, which was sensitive enough for distinguishing the patient sample from normal sample (the average concentration of AFP in healthy
A
human serum is no more than 25 ng/mL) with naked eye.[38] Fig. 5
The specificity of this enzymatic cascade reaction was evaluated using BSA,
human serum albumin (HSA), IgG, secretory immunoglobulin A (sIgA) and glucose (GO). As shown in Fig. S5, there was negligible absorbance change with separately adding 500 ng/mL of interfering substance, whereas the addition of 50 ng/mL AFP
give rise to an apparent absorbance change, implying the good performance for differentiating AFP from other potential interferents. Having demonstrated the sensitivity and selectivity of this proposed strategy, we next investigated the possibility of applying the immunoassay for AFP detection in normal human serum samples that taken from two healthy volunteers. The volunteers’
IP T
consent and approval from the Institutional Research Ethics Committee of Southwest University hospital were obtained for research purposes. As shown in Fig. S6, the
results obtained from our method were consistent with that obtained from commercial
SC R
HRP based ELISA kit, which guaranteed the feasible of the development for ALPlinked immunoassay.
U
4. Conclusion
In conclusion, the data reported here first pointed to that GTP exhibit an enzymatic
N
activity much the same as natural enzyme HRP, facilitating the oxidation of the
A
peroxidase substrate TMB to produce colour reaction by catalysing the decomposition
M
of H2O2 to generate •OH. Herein, we have made a further investigation and our results demonstrated that the peroxidase-like activity of GTP is derived from itself rather
ED
than acting as a reactant or hydrolysis-induced energy releasing. Kinetics assay showed that the peroxidase-like activity of GTP follows a typical Michaelis-Menten
PT
kinetics and GTP had a better catalytic efficiency than the reported small molecular enzyme-mimics. On the basis of this discovery, an enzymatic cascade reaction was
CC E
developed for quantitatively probing ALP activity, screening ALP inhibitor, and constructing immunoassay of AFP. We envision that our innovative research would somewhat cast a new light on the importance physiological functions of GTP and
A
encourage further exploration for design and development of enzymatic assays and immunoassays in biosensing. Acknowledgements
This work was supported by the research grants from National Natural Science Foundation of China (No.21405124, No.21175110) and Fundamental Research Funds for the Central Universities (No. XDJK2017D052).
References [1] L. Wang, Y. Liu, J. Li, Self-phosphorylating deoxyribozyme initiated cascade
IP T
enzymatic amplification for guanosine-5'-triphosphate detection, Anal. Chem. 86 (2014) 7907-7912.
[2] N. Wu, J. Lan, L. Yan, J. You, A sensitive colorimetric and fluorescent sensor based
SC R
on imidazolium-functionalized squaraines for the detection of GTP and alkaline phosphatase in aqueous solution, Chem. Commun. 50 (2014) 4438-4441.
[3] S. Shoji, S.E. Walker, K. Fredrick, Ribosomal Translocation: One Step Closer to the
U
Molecular Mechanism, Acs Chem. Biol. 4 (2009) 93-107.
N
[4] S.R. Neves, P.T. Ram, R. Iyengar, G protein pathways, Science 296(2002) 1636-9.
A
[5] J. Downward, Targeting RAS signalling pathways in cancer therapy, Nat. rev. Cancer 3 (2003) 11-22.
M
[6] A. Bianchi-Smiraglia, M.S. Rana, C.E. Foley, L.M. Paul, B.C. Lipchick, S.
ED
Moparthy, et al., Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution, Nat. Methods 14 (2017) 1003-1009. [7] H.R. Bourne, D.A. Sanders, F. Mccormick, The GTPase superfamily: a conserved
PT
switch for diverse cell functions, Nature 348 (1990) 125-132.
CC E
[8] D. Maity, M. Li, M. Ehlers, C. Schmuck, A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates, Chem. Commun. 53 (2016) 208-211.
[9] C. Yu, X. Chang, J. Liu, L. Ding, J. Peng, Y. Fang, Creation of reduced graphene
A
oxide based field effect transistors and their utilization in the detection and discrimination of nucleoside triphosphates, ACS Appl. Mater. Inter. 7 (2015) 1071810826. [10] J.E. Coleman, Structure and Mechanism of Alkaline Phosphatase, Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 441.
[11] J.L. Millan, Alkaline Phosphatases : Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes, Purinerg. Signal. 2 (2006) 335-241. [12] L.Y. Jin, Y.M. Dong, X.M. Wu, G.X. Cao, G.L. Wang, Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme, Anal. Chem. 87 (2015) 10429-10436.
IP T
[13] G.B. Jr, S. Ardakani, J. Ju, D. Jenkins, M.J. Cerelli, G.Y. Daniloff, et al.,
Monoclonal antibody assay for measuring bone-specific alkaline phosphatase activity
SC R
in serum, Clin. Chem. 41 (1995) 1560-1506.
[14] S. Sardiwal, P. Magnusson, D.J. Goldsmith, E.J. Lamb, Bone alkaline phosphatase in CKD-mineral bone disorder, Am. J. Kidney Dis. 62 (2013) 810-822.
U
[15] K. Ooi, K. Shiraki, Y. Morishita, T. Nobori, High-molecular intestinal alkaline
N
phosphatase in chronic liver diseases, J. Clin. Lab. Anal. 21 (2007) 133-139. [16] Wolf, L. Paul, Clinical significance of serum high-molecular-mass alkaline
A
phosphatase, alkaline phosphatase-lipoprotein-X complex, and intestinal variant
M
alkaline phosphatase, J. Clin. Lab. Anal. 8 (1994) 172-176. [17] G. Ramaswamy, V.R. Rao, L. Krishnamoorthy, G. Ramesh, R. Gomathy, D.
ED
Renukadevi, Serum levels of bone alkaline phosphatase in breast and prostate cancers with bone metastasis, Indian J. Clin. Biochem. 15 (2000) 110.
PT
[18] G.M. Rao, L.O. Morghom, Correlation between serum alkaline phosphatase activity and blood glucose levels, Enzyme 35 (1986) 57.
CC E
[19] D.M. Kemeny, S.J. Challacombe, ELISA and other solid phase immunoassays: theoretical and practical aspects, Febs Lett. 244 (1988) 503-504. [20] J. Sun, T. Hu, X. Xu, L. Wang, X. Yang, A fluorescent ELISA based on the enzyme-
A
triggered synthesis of poly(thymine)-templated copper nanoparticles, Nanoscale 8 (2016) 16846-16850. [21] Y. Lin, Y. Huang, J. Ren, X. Qu, Incorporating ATP into biomimetic catalysts for realizing exceptional enzymatic performance over a broad temperature range, NPG Asia Mater. 6 (2014) e114. [22] L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, et al., Intrinsic peroxidase-
like activity of ferromagnetic nanoparticles, Nat. Nanotechnol. 2 (2007) 577-583. [23] J. Tian, S. Liu, Y. Luo, X. Sun, Fe(iii)-based coordination polymernanoparticles: peroxidase-like catalytic activity and their application to hydrogen peroxide and glucose detection, Catal. Sci. Technol. 2 (2012) 432-436. [24] C. Xu, Z. Liu, L. Wu, J. Ren, X. Qu, Nucleoside Triphosphates as Promoters to Enhance Nanoceria Enzyme-like Activity and for Single-Nucleotide Polymorphism
IP T
Typing, Adv.Funct. Mater. 24 (2014) 1624-1630.
[25] J.R. Harbour, S.L. Issler, Involvement of the azide radical in the quenching of
SC R
singlet oxygen by azide anion in water, J. Am. Chem. Soc. 104 (1982) 903-905.
[26] A.P. Schaap, A.L. Thayer, G.R. Faler, K. Goda, T. Kimura, Singlet molecular oxygen and superoxide dismutase, J. Am. Chem. Soc. 91 (1974) 4025-4026.
U
[27] T. Lin, L. Zhong, L. Guo, F. Fu, G. Chen, Seeing diabetes: visual detection of
N
glucose based on the intrinsic peroxidase-like activity of MoS2 nanosheets, Nanoscale 6 (2014) 11856-11862.
A
[28] A. Qiao, C. Sun, D. Li, K. Xu, J. Guo, C. Wang, Peroxidase-like activity of
M
Fe3O4@Carbon nanoparticles enhances ascorbic acid-induced oxidative stress and selective damage to PC-3 prostate cancer cells, ACS Appl. Mater. Inter. 5 (2013) 13248-
ED
13257.
[29] L. Liu, Y. Shi, Y. Yang, M. Li, Y. Long, Y. Huang, et al., Fluorescein as an artificial
PT
enzyme to mimic peroxidase, Chem. Commun. 52 (2016) 13912-13915. [30] L. Liu, Y. Shi, M. Li, C. Sun, Y. Long, H. Zheng, Effect of carboxyl and amino
CC E
groups in fluorescein molecules on their peroxidase-like activity, Mol. Catal. 439 (2017) 186-192.
[31] M. Li, J. Yang, Y. Ou, Y. Shi, L. Liu, C. Sun, et al., Peroxidase-like activity of 2',7'-
A
difluorofluorescein and its application for galactose detection, Talanta 182 (2018) 422427. [32] M. Li, L. Liu, Y. Shi, Y. Yang, H. Zheng, Y. Long, Dichlorofluorescein as a peroxidase mimic and its application to glucose detection, New J. Chem. 41 (2017) 7578-7582. [33] Y. Shi, L. Liu, Y. Yu, Y. Long, H. Zheng, Acidic amino acids: A new-type of enzyme
mimics with application to biosensing and evaluating of antioxidant behaviour, Spectrochim. Acta A 201 (2018) 367-375. [34] S. U, P. D, P. R, Alkaline Phosphatase: An Overview, Indian J. Clin. Biochem. 29 (2014) 269-278. [35] N. Li, H. Ma, W. Cao, D. Wu, T. Yan, B. Du, et al., Highly sensitive electrochemical
N-doped graphene nanoribbons, Biosen. Bioelectro. 74 (2015) 786-791.
IP T
immunosensor for the detection of alpha fetoprotein based on PdNi nanoparticles and
[36] Y.W. Wang, L. Chen, M. Liang, H. Xu, S. Tang, H.H. Yang, et al., Sensitive
SC R
fluorescence immunoassay of alpha-fetoprotein through copper ions modulated growth of quantum dots in-situ, Sensor. Actuat. B-Chem. 247 (2017) 408-413.
[37] K. Sheng, W. Liu, L. Xu, Y. Jiang, X. Zhang, B. Dong, et al., An ultra-sensitive
U
label-free immunosensor toward alpha-fetoprotein detection based on three-
N
dimensional ordered IrOx inverse opals, Sensor. Actuat. B-Chem. 254 (2017) 660-668. [38] L. Liu, L. Tian, G. Zhao, Y. Huang, Q. Wei, W. Cao, Ultrasensitive electrochemical
A
immunosensor for alpha fetoprotein detection based on platinum nanoparticles
PT
Biographies
ED
Acta 986 (2017) 138-44.
M
anchored on cobalt oxide/graphene nanosheets for signal amplification, Anal. Chim.
Ying Shi is a doctoral student in School of Chemistry and Chemical Engineering,
CC E
Southwest University, China. Her major research interest is molecular spectrum analysis and optical biosensors construction.
A
Miao Yang received her M.S. degree in chemistry at Southwest University in 2016. Now she is a doctoral student in School of Chemistry and Chemical Engineering, Southwest University, China. Her major research interest is chemical theory and computation.
Li Liu received her Ph.D. degree in chemistry at Southwest University in 2017. Now she is a lecturer in College of Chemistry and Environmental Science at Qujing Normal University, China. Her research interests are sensors and spectrum analysis. Yanjiao Pang is master student in chemistry at Southwest University in 2016. Her major research interest is molecular spectrum analysis and biosensor construction.
IP T
Yijuan Long received her Ph.D. degree in chemistry at Southwest University in 2013. Now she is a senior experimentalist in School of Chemistry and Chemical
SC R
Engineering, Southwest University, China. Her research interests is spectrum analysis.
Huzhi Zheng received his Ph.D. degree in chemistry at Wuhan University in 2005.
U
Now he is a professor in School of Chemistry and Chemical Engineering, Southwest
ED
M
A
mimics and their application for biosensing.
N
University, China. His main research interests lie in drug delivery, novel enzyme
Figure and table captions
PT
Fig. 1. The peroxidase-like activity of GTP. (a) Schematic illustration and (b) absorption spectra of GTP-catalyzed oxidation of TMB by H2O2 to form oxTMB in
CC E
pH 5.0 PBS buffer. (c) Absorbance value changes with different concentrations of GTP-catalyzed oxidation of TMB in the presence of H2O2. Fig. 2. Production of hydroxyl radicals catalyzed by GTP. (a) The catalytic reaction in
A
the presence of different concentration of radical scavengers. (b) ESR spectra of H2O2/DMPO spin adduct produced in PBS buffer (pH 5.0) containing 50 mM DMPO, 10 mM H2O2, and various concentrations of catalyst. Fig. 3. Steady-state kinetic assays of GTP. (a) The concentration of H2O2 was 10 mM and the TMB concentration was varied. (b) The concentration of TMB was 0.80 mM
and the H2O2 concentration was varied. Error bars represent the standard deviations of three independent experiments. Fig. 4. ALP sensing based on GTP-mediated enzyme cascade reaction. (a) Principle illustration of the colorimetric sensor for ALP detection. (b) Absorption spectra of GTP-catalyzed TMB-H2O2 system in the presence of ALP with different units (0,
IP T
0.01, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 100, 150, 200 U/L). (c) Calibration curve for ALP detection. (d) The linearity of logarithmic absorbance changes with respect to
logarithmic ALP activity. (e) The selectivity of the GTP-based method for ALP assay.
SC R
A0 represents the reaction without ALP and A represents the reaction with different ALP levels.
U
Fig. 5. AFP detection based on GTP-mediated enzyme cascade reaction. (a)
Schematic illustration of the working principle for AFP. (b) Photographs and (c) the
N
corresponding absorption spectra of the ALP-linked immunoassay integrating with
A
the peroxidase-like activity of GTP in the presence of different amounts of AFP (0,
M
0.5, 1, 5, 10, 20, 30, 50, 100, 150 ng/mL). (d) Calibration curve for AFP detection. Error bars were estimated from three replicate measurements. A0 represents the
ED
reaction without AFP and A represents the reaction with different concentrations of AFP.
PT
Table1. Comparison of the apparent Michaelis-Menten constant and maximal
A
CC E
velocity of GTP, other small moleculer and HRP.
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-1
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-2
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-3
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-4
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-5
Table1. Comparison of the apparent Michaelis-Menten constant and maximal velocity of GTP, other small moleculer, and HRP.
1.0×10-5
F[29]
1.0×10-4
CF[30]
1.0×10-4
AF[30]
1.0×10-4
DCF[32]
1.0×10-4
DFF[31]
1.0×10-4
L-Glu[33]
1.0×10-2
L-Asp[33]
1.0×10-2
HRP[33]
2.3×10-11
Km (mM) 2.93±0.219 0.761±0.001 1.31±0.129 1.11±0.044 0.304±0.026 1.66 ± 0.042 1.90 ± 0.126 1.86 ± 0.232 0.21 0.06 1.78 2.97 0.6268±0.219 0.0144±0.002 0.4370±0.083 0.0115±0.001 0.179±0.020 1.18±0.147
N A M ED PT CC E A
Vmax (10-8 M s-1) 9.15±0.052 1.80±0.002 0.418 ± 0.11 0.438 ± 0.18 0.547 ± 0.10 0.836 ± 0.12 0.384 ± 0.16 0.217 ± 0.02 0.207 0.096 0.404 0.983 0.2287±0.052 0.1927±0.006 0.3560±0.040 0.2713±0.003 3.895±0.218 7.307 ± 0.824
kcat (s-1) [a] 9.15×10-3 1.80×10-3 4.18×10-5 4.38×10-5 5.47×10-5 8.36×10-5 3.84×10-5 2.17×10-5 2.07×10-5 0.96×10-5 4.04×10-5 9.83×10-5 2.29×10-7 1.93×10-7 3.56×10-7 2.71×10-7 1.69×103 3.18×103
IP T
GTP
Substrate TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2
SC R
[E] (M)
U
Catalyst
S0925-4005(18)31469-2 https://doi.org/10.1016/j.snb.2018.08.038 SNB 25185
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
27-3-2018 3-8-2018 7-8-2018
Please cite this article as: Shi Y, Yang M, Liu L, Pang Y, Long Y, Zheng H, GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.08.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay
IP T
Ying Shi1, Miao Yang1, Li Liu1,2, Yanjiao Pang1, Yijuan Long1, Huzhi Zheng1,* 1
SC R
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest
University), Ministry of Education, College of Chemistry and Chemical Engineering
U
Southwest University Beibei, Chongqing, 400715, P. R. China.
2
N
College of Chemistry and Environmental Science Qujing Normal University Qujing,
A
655011, P. R. China.
M
*Corresponding author: Huzhi Zheng, Email: [email protected]
PT
ED
Graphical Abstract
We showed for the first time that GTP exhibited peroxidase-like activity and a
CC E
colorimetric platform for ALP and AFP detection was designed based on the enzymatic cascade reaction by coupling ALP-catalyzed dephosphorylation of GTP
A
with GTP-catalyzed oxidation of TMB.
IP T SC R U
Highlights
GTP was first found to exhibit an intrinsic peroxidase-like activity.
The peroxidase-like activity of GTP was closely related to the phosphate group.
A sensitive colorimetric platform was designed for ALP and AFP sensing based on enzymatic cascade reaction.
This finding provides further insights into new biological function of GTP and construction of biosensors based on the enzyme-like activity of GTP.
A
CC E
PT
ED
M
A
N
Abstract: Guanosine triphosphate (GTP), a substrate for RNA synthesis, plays a pivotal role in life-form metabolism at the cellular level. Herein, we first showed that GTP exhibited an intrinsic peroxidase-mimic activity, accelerating H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a color reaction. Compared to GTP, weaker catalytic activity was observed from guanosine diphosphate (GDP) and virtually no catalytic activity was observed from guanosine monophosphate
IP T
(GMP) and guanosine. Alkaline phosphatase (ALP) can catalyze the dephosphorylation
of GTP with in situ generation of GDP, GMP and guanosine, which followed by
SC R
receding the oxidation of TMB. Inspired by this, a novel colorimetric sensor for ALP
detection was designed based on the enzymatic cascade reaction by coupling ALPcatalyzed dephosphorylation of GTP with GTP-catalyzed oxidation of TMB. This
U
method allowed ALP sensing in a range from 0.01 to 100 U/L with a detection limit of
N
0.009 U/L, which was sensitive enough for ALP activity assay in biological samples
A
(the normal ALP level range of adult serum is 20-140 U/L). Moreover, ALP is widely used in enzyme-linked immunosorbent assays (ELISA) as a signal reporter, the proof-
M
of-concept ELISA for alpha-fetoprotein (AFP) detection was established. This
ED
unexpected discovery not only extends the new biological function of GTP, but also holds great promise for development of versatile biosensors that initiate the
PT
concentration changes of GTP.
A
CC E
Keywords: Guanosine triphosphate; Peroxidase-like activity; Alkaline phosphatase; Alpha-fetoprotein; ELISA
1. Introduction Guanosine triphosphate (GTP), well-known as a member of fundamental building blocks for synthesis of RNA during the transcription process, plays a key role in the regulation of a large array of cellular process and biochemical pathway.[1, 2] For example, GTP can act as a source of energy like adenosine triphosphate (ATP) in the course of biological metabolism, but it more specific for protein synthesis.[3] GTP
IP T
also has the role in signal transduction with G-proteins, during which GTP is
converted to guanosine diphosphate (GDP) through the action of GTPases that
SC R
relating to a variety of human diseases such as cancer.[4-7] In addition, GTP is
involved in citric acid cycle referred to energy transfer in the cell metabolism.[8, 9] However, despite its orthodox role in life activities, experimental attempts to observe
U
the new biological function of GTP remain sparse.
N
Alkaline phosphatase (ALP, EC 3.1.3.1), widespread in liver, bone and kidney, is a non-specific hydrolase that catalyses the dephosphorylation process of broad substrates
A
including proteins, nucleic acids and small molecules that contain phosphate functional
M
groups in alkali condition.[10, 11] It plays a significant role in regulating phosphate metabolism involved in cell cycle, growth, apoptosis and signal transduction
ED
pathways in biological system.[12] The abnormal expression level of ALP in serum can be identified as a diagnostic indicator for a series of diseases such as bone diseases
PT
(osteoblastic bone cancer, Paget’s disease, ),[13, 14] liver dysfunction (cancer, hepatitis, and obstructive jaundice),[15, 16] breast and prostatic cancer,[17] and diabetes[18] etc.
CC E
Moreover, despite the physiological functions of ALP in related biological processes, it is also commonly used as an enzyme-label within enzyme-linked immunosorbent assays (ELISA) for protein biomarker detection owing to being easily conjugated to
A
antibodies, inexpensive, broad substrate specificity and commercial availability and high catalytic activity.[12, 19, 20] Here, in the present work we first demonstrated that GTP exhibited the catalytical activity like nature horseradish peroxidase (HRP), catalysing the oxidation of the peroxidase substrates 3,3′,5,5′-tetramethylbenzidine (TMB) to produce colour reaction in the presence of H2O2. Following a series of experiments, we sought to investigate
and prove this intriguingly discovery. The peroxidase-like activity of GTP is indeed attributed to GTP itself as a catalyst rather than a reactant during the reaction. Meanwhile, experiments were also extended to GTP analogue, i.e., guanosine diphosphate (GDP), guanosine monophosphate (GMP) and guanosine. The results demonstrated that the peroxidase-like activity of GTP and its analogue were depended on the phosphate group and the catalytic ability rapidly weakened or even lost along
IP T
with the decrease of phosphate group: weaker catalytic activity was observed from guanosine diphosphate (GDP) and virtually no catalytic activity was observed from
SC R
guanosine monophosphate (GMP) and guanosine under the same condition.
Inspired by this significant finding, a colorimetric platform for ALP detection was designed based on GTP-mediated enzymatic cascade reaction. Upon addition of ALP,
U
GTP can be hydrolyzed to generate the product GDP with weaker catalytic activity or
N
GMP and guanosine with no catalytic activity, and thereby leading to the declined readout signal of oxidized TMB (oxTMB). Then, as a proof of concept, ALP-labelled
A
enzyme-linked immunosorbent assay for alpha fetoprotein (AFP) was designed
M
coupling with the biocatalytic activity of GTP. The proposed immunoassay has been applied for AFP level detection in serum samples with satisfied results. This significant
ED
discovery not only extends the new biological function of GTP, but also opens new opportunities to impel the development of biosensors with triggering the variation of
PT
GTP concentration.
CC E
2. Material and methods 2.1. Chemicals
Guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine
A
monophosphate (GMP), guanosine (99%) and 3,3′,5,5′-tetramethylbenzidine (TMB) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Alkaline phosphtase (ALP) was purchased from Sigma-Aldrich (St. Louis, MO). Human AFP antigen and antibody were purchased from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Hydrogen peroxide (H2O2) was obtained from Chongqing Chuandong
Chemical Co., Ltd. (Chongqing, China). All the commercial available regents were used without further purification. All solutions were prepared using ultrapure water (18.2 MΩ·cm-1) from a Milli-Q automatic ultrapure water system. 2.2. Peroxidase activity assays GTP to functionally mimic peroxidase was evaluated by the catalytic oxidation of
IP T
organic substrate TMB. Briefly, 100 μL of 100 μM GTP, 100 μL of 8.0 mM organic
peroxidase substrates and 100 μL of 100 mM H2O2 were added into pH 5.0 phosphate
SC R
buffer solution (PBS, 20 mM) to a volume of 1.0 mL to produce color product. After incubation under the optimal condition, the absorption spectra of the solution were recorded on a UV-2450 spectrometer (Shimadzu, Japan).
U
2.3. Electron spin resonance spectroscopy
N
Electron spin resonance spectroscopy (ESR) was used to detect hydroxyl radical
A
(•OH) generation with spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The
M
reaction mixture was composed of 50 mM DMPO, 10 mM H2O2, and different concentration of GTP in pH 5.0 PBS. Data collection began after sample mixing for 1
ED
min and the ESR spectra were recorded on a Bruker ESR 300E using the following settings: 20 mW microwave power, 1 G field modulation, and 100 G scan range.
PT
2.4. GTP hydrolysis and free inorganic phosphate determination GTP hydrolysis degree was measured using a malachite green-ammonium
CC E
molybdate assay.[21] A typical procedure was carried out as follows: (1) 500 μL of GTP (10 μM) was incubated at different temperatures for 40 min with the solution at 4°C as a blank. (2) Malachite green (0.081%), polyvinyl alcohol (2.3%), ammonium
A
heptamolybdate tetrahydrate (5.7% in 6 M HCl) and water were mixed in a ratio of 2:1:1:2. (3) Subsequently, 100 μL of the above obtained mixture solution was dropped into the GTP solution with adding 40 μL of 3.4% sodium citrate to halt the hydrolysis of GTP. (4) Then the reaction was allowed to proceed for 15 min and the absorbance values at 620 nm were recorded. In addition, a standard curve for phosphate was
obtained using different NaH2PO4 concentrations, and the amount of inorganic phosphate generated by GTP hydrolysis was calculated using the standard curve. 2.5. Ultra-performance liquid chromatography-mass spectrum (UPLC-MS) Analysis Ultra-performance liquid chromatography (UPLC) was carried out on an Acquity
IP T
UPLC system (Waters, UK). Separation was performed on an Acquity BEH C18
column (2.1 × 50 mm, 1.7 μm particle size) with column oven temperature at 40°C.
SC R
The mobile phases were: 80% acetonitrile and 20% methanol. The flow rate was 0.2 mL/min. Separated substances were detected by Xevo TQ-S mass spectrometer
(Waters, UK) with an electrospray ionization source. Source conditions were fixed as
U
follows: source temperature, 150°C; desolvation temperature, 350°C; cone voltage, 35
N
eV; capillary voltage, 2.5 kV.
A
2.6. Kinetic analysis
M
Steady-state kinetic assays for the catalytic oxidation of TMB by GTP were carried out by monitoring the absorbance change at 652 nm at selected time intervals.
ED
Experiments were carried out using 10 μM GTP at the optimal temperature in a reaction volume of 4.0 mL pH 5.0 PBS by varying concentrations of TMB at a fixed
PT
concentration of H2O2 or vice versa. The kinetic parameters were calculated based on Michaelis-Menten equation: v = Vmax•[S] /([S]+ Km), where v is the initial velocity,
CC E
Vmax is the maximal reaction velocity, and [S] is the concentration of substrate and Km is the Michaelis constant.
A
2.7. ALP detection The ALP activity assay was conducted based on its enzymatic hydrolysis of GTP to
generate GDP, GMP and guanosine. First, 10 μL of ALP with different activities ranging from 0 to 200 U/L, 10 μL of 100 μM GTP was added into 30 μL of 10 mM pH 8.0 Tris-HCl buffer (containing 5 mM MgCl2 and 0.1 mM ZnCl2) for an incubation time of 1 h at 40°C. After that, 100 μL of 8.0 mM TMB, 100 μL of 100
mM H2O2, and 100 μL of pH 5.0 PBS were added in the above solution and diluted to 1.0 mL. Finally, the mixture was incubated at 50°C for 40 min and the absorbance of the oxTMB at 652 nm was recorded. 2.8. Immunoassay for AFP First, 100 µL of 0.50 μg/mL capture antibody (Ab1) dissolved in 0.050 M NaHCO3-
IP T
Na2CO3 buffer (pH 9.6) was added to 96-well microplates and incubated at 4°C
overnight followed by rinsing with the washing buffer (0.010 M PBS containing
SC R
0.05% Tween 20, pH 7.4) for five times. Then the wells were blocked with 100 μL
1% bovine serum albumin (BSA) at 37°C for 1 h and washed with the washing buffer thoroughly. Following that, 100 μL of AFP with different concentrations were added
U
into the wells and incubated at 37°C for 90 min followed by washing. Subsequently, 100 μL of 2.0 μg/mL biotinylated-antibody (Ab2) were added into each well for an
N
incubation time of 30 min at 37°C and then washed five times with washing buffer.
A
Then 100 μL of avidin-ALP (1:500) were added to conjugate to antibody through
M
biotin-avidin reaction for an incubation time of 30 min at 37°C and again washed thoroughly. After the formation of sandwich structure, 20 μL of 15 μM GTP in 10
ED
mM Tris-HCl buffer (pH 8.0) containing 5 mM MgCl2 and 0.1 mM ZnCl2 was added and incubated at 40°C for 60 min. Finally, 30 μL of 8.0 mM TMB, 30 μL of 100 mM
PT
H2O2, 30 μL of pH 3.0 PBS buffer, and 190 μL of ultrapure water were added into the 96 well plates and reacted at 50°C for 40 min before the absorbance of oxTMB at 652
CC E
nm was measured.
3. Results and discussion
A
3.1. Peroxidase-like properties of GTP GTP could apply as a catalyst for the oxidation of peroxidase substrates TMB to
oxidized TMB (oxTMB) in the presence of H2O2 (Fig. 1a). As shown in Fig. 1b, a characteristic absorbance of oxTMB at 652 nm can be observed upon adding GTP into TMB-H2O2 system, while there was negligible absorbance variation in the absence of GTP, TMB, or H2O2. Fig. 1c displayed that the increasing concentration of GTP could
improve the reaction rates, reaching a plateau at about 10 μM. These results demonstrated that GTP can function as peroxidase mimics. Meanwhile, we found that the catalytic efficiency of GTP, like HRP, dependent on pH, temperature, H2O2 concentrations and incubation time.[22] As shown in Fig. S1, the optimal pH, temperature, H2O2 concentration and reaction time for catalytic activity of GTP were evaluated to be 5.0, 50°C, 10 mM and 40 min, respectively. Note that GTP
IP T
required a high H2O2 concentration to reach the maximum level of peroxidase-like activity and then remain stable with increased H2O2 concentration while natural enzyme
SC R
HRP was denatured at high H2O2 concentration, suggesting that the catalytic activity of GTP exhibited higher tolerance level at high H2O2 concentration than HRP. [22, 23] Fig. 1
U
3.2. Activity is due to intact GTP not hidrolysis releasing energy
N
Qu et al. reported that GTP can improve the oxidase-like activity of nanoceria due
A
to the hydrolysis of GTP coupling with releasing energy.[24] It is important to
M
exclude that the hydrolysis of GTP render its ability to promote the TMB-H2O2 reaction. Then hydrolysis degree was evaluated by measuring the amount of inorganic
ED
phosphate released using a malachite green-ammonium molybdate assay.[21] As shown in Fig. S2a, the hydrolysis amounts of GTP taken place under our experimental
PT
temperature were no more than 0.55 μM, that is, hydrolysis degree could not exceed 5.5%, suggesting that little energy would release and this change in the reaction was
CC E
insignificant.
UPLC-MS was conducted to further ascertain the integrity of GTP during the
reaction process, i. e., GTP can act as catalyst to accelerate the TMB-H2O2 reaction.
A
As shown in Fig. S2b, the curve 1, a single peak (fragment ion at m/z 158.86) with retention time at 0.92 min, display the UPLC-MS analysis of GTP in the absence of TMB and H2O2 as a control. Curve 2 illustrates the separation result of GTP mediated TMB-H2O2 reaction. For the two peaks observed, one with the same retention time (0.91 min) corresponds to GTP and another peak (fragment ion at m/z 195.11) with
retention time at 3.8 min corresponds to oxTMB. In addition, the peak area of GTP in curve 2 (32555) was almost identical to that in curve 1 (31345). The sum of evidences pointed to that GTP changed little in either quality or quantity during the reaction and supported that GTP indeed act as a catalyst to mimic peroxidase. 3.3. Production of hydroxyl radicals catalyzed by GTP
IP T
Formation of reactive oxygen species (ROS) is of vital importance in catalytic oxidation chemistry. To identify the kind of ROS formation catalyzed by GTP,
SC R
different radical scavengers including ascorbic acid (AA), sodium azide (NaN3) and superoxide dismutase (SOD) were introduced to GTP-TMB-H2O2 system. As
illustrated in Fig. 2a, little variation in the absorbance of oxTMB at 652 nm was caused with addition of varied concentration of NaN3 and SOD, which were used as
U
scavengers for 1O2 and O2•−, respectively.[25,26] Whereas, the absorbance values
N
decreased progressively with the addition of increased concentration of AA, an
A
effective •OH and O2•− scavengers.[27] These results demonstrated that •OH was
M
generated during the GTP mediated-catalytic reaction.
ED
Electron spin resonance (ESR) is a powerful and direct technique for ROS qualitative analysis using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. In the presence of •OH, DMPO can capture the short-lived •OH to form the DMPO/•OH
PT
spin adduct, which is a relatively stable paramagnetic product with a characteristic ESR signal of a 1:2:2:1 quartet.[28] As shown in Fig. 2b, H2O2 added alone showed
CC E
poor DMPO/•OH adduct signal intensity, but the characteristic signal was enhanced by adding GTP and found to be progressively increased with the increasing concentration, suggesting that GTP possessed the peroxidase-mimic ability to
A
decompose H2O2 to generate reactive •OH radicals. Fig. 2
3.4. Kinetics assay The peroxidase-like catalytic mechanism and kinetic parameters of GTP were further investigated using steady-state kinetics. As shown in Fig. 3, the oxidation
reaction process catalyzed by GTP could be modeled phenomenologically following the conventional enzymatic dynamic regulation of the Michiaelis-Menten equation v = Vmax•[S] /([S]+ Km), where v is the initial velocity, Vmax is the maximal reaction velocity, Km is the Michaelis constant and [S] is the concentration of the substrate. We also compared the kinetic parameters of GTP with other reported small molecular enzyme-mimic: fluorescein (F)[29] and its derivatives including 5(6)-
IP T
carboxyfluorescein (CF), 5(6)-aminofluorescein (AF)[30], 2', 7'-difluorofluorescein (DFF) [31] and 2′,7′- dichlorofluorescein (DCF)[32] as well as acidic amino acids
SC R
including L-glutamic acid (L-Glu) and L-aspartic acid (L-Asp)[33]. As summarized in Table 1, the apparent Km value, indicating the binding affinity between enzyme and substrates, of GTP toward H2O2 was lower than that of F and its derivatives except for
U
DCF and acidic amino acids, suggesting that GTP had a better affinity to H2O2. The
N
Vmax value of GTP was higher and the catalytic constant kcat of GTP was two orders of
A
magnitude higher that of fluorescein and its derivatives and four orders of magnitude higher than that of acidic amino acid. From these observations it is evident that the
M
catalytic activity of GTP is more efficient than other small molecular enzyme-mimics.
ED
However, the kcat value of GTP is six orders of magnitude lower than that of natural enzyme HRP. Thus, it is still a great challenge to design and screen small molecular
Fig. 3 Table 1
CC E
PT
mimetic enzymes with higher catalytic activity.
3.5. ALP sensing based on peroxidase-like GTP ALP, an essential enzyme in phosphate metabolism, can catalyze the hydrolysis of
A
phosphoryl eaters and the abnormal expression level was associated with several diseases.[12] GTP as one of the typical substrates of ALP can be hydrolyzed to yield GDP, GMP and guanosine phosphate[2] and we envisioned that ALP can be detected based on the peroxidase-like activity of GTP. To verify the feasibility of this assay, we evaluated that whether GDP, GMP and guanosine could catalyze the oxidation of TMB in the presence of H2O2 under the same experiment condition. As shown in Fig.
S3, only GDP possessed the peroxidase-like activity but one time lower than GTP, i.e., the catalytic ability rapidly weakened or even lost along with the decrease of phosphate group. These observations fully supported that ALP activity sensing platform can be accordingly constructed based on the significance level of difference between GTP and its hydrolysis product.
IP T
The response progress of GTP-mediated enzymatic cascade reaction for ALP detection was illustrated in Fig. 4a, upon addition of ALP, GTP with high catalytic activity was hydrolyzed to generate GDP with weaker catalytic activity and GMP
SC R
guanosine non-catalytic activity to further regulate the readout signal of oxidized
TMB. The absorption spectra were recorded by varying ALP concentration from 0.01 to 200 U/L and the absorbance decreased gradually upon enhancing ALP level as a
U
result of the consumption of GTP (Fig. 4b). The relationship between the absorbance
N
changes of this colorimetric sensor and the ALP activity was depicted in Fig. 4c, the
A
absorbance changes was plotted as a function of ALP level in a range from 0.01-100
M
U/L (Fig. 4d) with a detection limit of 0.009 U/L, which was sensitive enough for ALP activity assay in biological samples (the normal ALP level range of adult serum
ED
is 20-140 U/L).[34]
Then the selectivity of this GTP-based ALP sensing platform was evaluated by
PT
separated addition of several proteins including bovine serum albumin (BSA), lysozyme, glucose oxidase (GOx), uricase, immunoglobulin G (IgG), trypsin and
CC E
esterase under the same condition. The concentration was 200 U/L for ALP and 0.10 mg/mL for each other interfering proteins. As shown in Fig. 4e, all the interference proteins showed no hydrolysis on GTP and the peroxidase activity of GTP was
A
maintained, demonstrating the high selectivity of the proposed sensing approach. Fig. 4
Given the importance of screening enzyme regulators in clinical chemistry and drug development, Na3VO4, a common ALP inhibitor was selected for the inhibiting assay.[12] As shown in Figure S4, the ALP activity decreased with the addition of increased Na3VO4 concentration and the IC50 value (half-maximal inhibitory
concentration) for 500 U/L ALP was determined to be 35 μM, demonstrating the feasibility of this protocol for screening ALP inhibitor in drug discovery. 3.6. ALP-linked immunoassay for AFP Considering the significance of ALP as the most commonly enzyme label in enzyme-linked immunosorbent assays due to its high catalytic activity, broad
IP T
substrate specificity and commercial availability,[12] ALP-based immunoassay
integrating with the peroxidase-like activity of GTP was established. In this study,
SC R
AFP, a well-known biomarker of some cancerous diseases in clinical diagnosis,[3537] was used as a model analyte in our proof-of-concept experiment. As
schematically illustrated in Fig. 5a, capture antibody (Ab1) was immobilized on the
U
microtiter plates to recognize target antigen AFP, then biotinylated-antibody (Ab2) was further conjugated and a sandwich structure can be formed. Subsequently, ALP
N
can be anchored on the sandwich structure through the specific reaction between
A
biotin and avidin and further causing the depletion of GTP due to hydrolysis of
M
labeled ALP, which eventually leading to weaken the oxidation of TMB. As shown in Fig. 5c, the absorbance gradually decreased with the target antigen AFP concentration
ED
rising and a linear relationship can be obtained in the range from 1.0-100 ng/mL with a detection limit of 0.5 ng/mL (Fig. 5d). The color responses to different
PT
concentrations of AFP can be seen from the photos in Fig. 5b, an obvious color changed can be observed with naked eye compared to the blank color when the
CC E
concentration of AFP was 20 ng/mL, which was sensitive enough for distinguishing the patient sample from normal sample (the average concentration of AFP in healthy
A
human serum is no more than 25 ng/mL) with naked eye.[38] Fig. 5
The specificity of this enzymatic cascade reaction was evaluated using BSA,
human serum albumin (HSA), IgG, secretory immunoglobulin A (sIgA) and glucose (GO). As shown in Fig. S5, there was negligible absorbance change with separately adding 500 ng/mL of interfering substance, whereas the addition of 50 ng/mL AFP
give rise to an apparent absorbance change, implying the good performance for differentiating AFP from other potential interferents. Having demonstrated the sensitivity and selectivity of this proposed strategy, we next investigated the possibility of applying the immunoassay for AFP detection in normal human serum samples that taken from two healthy volunteers. The volunteers’
IP T
consent and approval from the Institutional Research Ethics Committee of Southwest University hospital were obtained for research purposes. As shown in Fig. S6, the
results obtained from our method were consistent with that obtained from commercial
SC R
HRP based ELISA kit, which guaranteed the feasible of the development for ALPlinked immunoassay.
U
4. Conclusion
In conclusion, the data reported here first pointed to that GTP exhibit an enzymatic
N
activity much the same as natural enzyme HRP, facilitating the oxidation of the
A
peroxidase substrate TMB to produce colour reaction by catalysing the decomposition
M
of H2O2 to generate •OH. Herein, we have made a further investigation and our results demonstrated that the peroxidase-like activity of GTP is derived from itself rather
ED
than acting as a reactant or hydrolysis-induced energy releasing. Kinetics assay showed that the peroxidase-like activity of GTP follows a typical Michaelis-Menten
PT
kinetics and GTP had a better catalytic efficiency than the reported small molecular enzyme-mimics. On the basis of this discovery, an enzymatic cascade reaction was
CC E
developed for quantitatively probing ALP activity, screening ALP inhibitor, and constructing immunoassay of AFP. We envision that our innovative research would somewhat cast a new light on the importance physiological functions of GTP and
A
encourage further exploration for design and development of enzymatic assays and immunoassays in biosensing. Acknowledgements
This work was supported by the research grants from National Natural Science Foundation of China (No.21405124, No.21175110) and Fundamental Research Funds for the Central Universities (No. XDJK2017D052).
References [1] L. Wang, Y. Liu, J. Li, Self-phosphorylating deoxyribozyme initiated cascade
IP T
enzymatic amplification for guanosine-5'-triphosphate detection, Anal. Chem. 86 (2014) 7907-7912.
[2] N. Wu, J. Lan, L. Yan, J. You, A sensitive colorimetric and fluorescent sensor based
SC R
on imidazolium-functionalized squaraines for the detection of GTP and alkaline phosphatase in aqueous solution, Chem. Commun. 50 (2014) 4438-4441.
[3] S. Shoji, S.E. Walker, K. Fredrick, Ribosomal Translocation: One Step Closer to the
U
Molecular Mechanism, Acs Chem. Biol. 4 (2009) 93-107.
N
[4] S.R. Neves, P.T. Ram, R. Iyengar, G protein pathways, Science 296(2002) 1636-9.
A
[5] J. Downward, Targeting RAS signalling pathways in cancer therapy, Nat. rev. Cancer 3 (2003) 11-22.
M
[6] A. Bianchi-Smiraglia, M.S. Rana, C.E. Foley, L.M. Paul, B.C. Lipchick, S.
ED
Moparthy, et al., Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution, Nat. Methods 14 (2017) 1003-1009. [7] H.R. Bourne, D.A. Sanders, F. Mccormick, The GTPase superfamily: a conserved
PT
switch for diverse cell functions, Nature 348 (1990) 125-132.
CC E
[8] D. Maity, M. Li, M. Ehlers, C. Schmuck, A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates, Chem. Commun. 53 (2016) 208-211.
[9] C. Yu, X. Chang, J. Liu, L. Ding, J. Peng, Y. Fang, Creation of reduced graphene
A
oxide based field effect transistors and their utilization in the detection and discrimination of nucleoside triphosphates, ACS Appl. Mater. Inter. 7 (2015) 1071810826. [10] J.E. Coleman, Structure and Mechanism of Alkaline Phosphatase, Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 441.
[11] J.L. Millan, Alkaline Phosphatases : Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes, Purinerg. Signal. 2 (2006) 335-241. [12] L.Y. Jin, Y.M. Dong, X.M. Wu, G.X. Cao, G.L. Wang, Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme, Anal. Chem. 87 (2015) 10429-10436.
IP T
[13] G.B. Jr, S. Ardakani, J. Ju, D. Jenkins, M.J. Cerelli, G.Y. Daniloff, et al.,
Monoclonal antibody assay for measuring bone-specific alkaline phosphatase activity
SC R
in serum, Clin. Chem. 41 (1995) 1560-1506.
[14] S. Sardiwal, P. Magnusson, D.J. Goldsmith, E.J. Lamb, Bone alkaline phosphatase in CKD-mineral bone disorder, Am. J. Kidney Dis. 62 (2013) 810-822.
U
[15] K. Ooi, K. Shiraki, Y. Morishita, T. Nobori, High-molecular intestinal alkaline
N
phosphatase in chronic liver diseases, J. Clin. Lab. Anal. 21 (2007) 133-139. [16] Wolf, L. Paul, Clinical significance of serum high-molecular-mass alkaline
A
phosphatase, alkaline phosphatase-lipoprotein-X complex, and intestinal variant
M
alkaline phosphatase, J. Clin. Lab. Anal. 8 (1994) 172-176. [17] G. Ramaswamy, V.R. Rao, L. Krishnamoorthy, G. Ramesh, R. Gomathy, D.
ED
Renukadevi, Serum levels of bone alkaline phosphatase in breast and prostate cancers with bone metastasis, Indian J. Clin. Biochem. 15 (2000) 110.
PT
[18] G.M. Rao, L.O. Morghom, Correlation between serum alkaline phosphatase activity and blood glucose levels, Enzyme 35 (1986) 57.
CC E
[19] D.M. Kemeny, S.J. Challacombe, ELISA and other solid phase immunoassays: theoretical and practical aspects, Febs Lett. 244 (1988) 503-504. [20] J. Sun, T. Hu, X. Xu, L. Wang, X. Yang, A fluorescent ELISA based on the enzyme-
A
triggered synthesis of poly(thymine)-templated copper nanoparticles, Nanoscale 8 (2016) 16846-16850. [21] Y. Lin, Y. Huang, J. Ren, X. Qu, Incorporating ATP into biomimetic catalysts for realizing exceptional enzymatic performance over a broad temperature range, NPG Asia Mater. 6 (2014) e114. [22] L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, et al., Intrinsic peroxidase-
like activity of ferromagnetic nanoparticles, Nat. Nanotechnol. 2 (2007) 577-583. [23] J. Tian, S. Liu, Y. Luo, X. Sun, Fe(iii)-based coordination polymernanoparticles: peroxidase-like catalytic activity and their application to hydrogen peroxide and glucose detection, Catal. Sci. Technol. 2 (2012) 432-436. [24] C. Xu, Z. Liu, L. Wu, J. Ren, X. Qu, Nucleoside Triphosphates as Promoters to Enhance Nanoceria Enzyme-like Activity and for Single-Nucleotide Polymorphism
IP T
Typing, Adv.Funct. Mater. 24 (2014) 1624-1630.
[25] J.R. Harbour, S.L. Issler, Involvement of the azide radical in the quenching of
SC R
singlet oxygen by azide anion in water, J. Am. Chem. Soc. 104 (1982) 903-905.
[26] A.P. Schaap, A.L. Thayer, G.R. Faler, K. Goda, T. Kimura, Singlet molecular oxygen and superoxide dismutase, J. Am. Chem. Soc. 91 (1974) 4025-4026.
U
[27] T. Lin, L. Zhong, L. Guo, F. Fu, G. Chen, Seeing diabetes: visual detection of
N
glucose based on the intrinsic peroxidase-like activity of MoS2 nanosheets, Nanoscale 6 (2014) 11856-11862.
A
[28] A. Qiao, C. Sun, D. Li, K. Xu, J. Guo, C. Wang, Peroxidase-like activity of
M
Fe3O4@Carbon nanoparticles enhances ascorbic acid-induced oxidative stress and selective damage to PC-3 prostate cancer cells, ACS Appl. Mater. Inter. 5 (2013) 13248-
ED
13257.
[29] L. Liu, Y. Shi, Y. Yang, M. Li, Y. Long, Y. Huang, et al., Fluorescein as an artificial
PT
enzyme to mimic peroxidase, Chem. Commun. 52 (2016) 13912-13915. [30] L. Liu, Y. Shi, M. Li, C. Sun, Y. Long, H. Zheng, Effect of carboxyl and amino
CC E
groups in fluorescein molecules on their peroxidase-like activity, Mol. Catal. 439 (2017) 186-192.
[31] M. Li, J. Yang, Y. Ou, Y. Shi, L. Liu, C. Sun, et al., Peroxidase-like activity of 2',7'-
A
difluorofluorescein and its application for galactose detection, Talanta 182 (2018) 422427. [32] M. Li, L. Liu, Y. Shi, Y. Yang, H. Zheng, Y. Long, Dichlorofluorescein as a peroxidase mimic and its application to glucose detection, New J. Chem. 41 (2017) 7578-7582. [33] Y. Shi, L. Liu, Y. Yu, Y. Long, H. Zheng, Acidic amino acids: A new-type of enzyme
mimics with application to biosensing and evaluating of antioxidant behaviour, Spectrochim. Acta A 201 (2018) 367-375. [34] S. U, P. D, P. R, Alkaline Phosphatase: An Overview, Indian J. Clin. Biochem. 29 (2014) 269-278. [35] N. Li, H. Ma, W. Cao, D. Wu, T. Yan, B. Du, et al., Highly sensitive electrochemical
N-doped graphene nanoribbons, Biosen. Bioelectro. 74 (2015) 786-791.
IP T
immunosensor for the detection of alpha fetoprotein based on PdNi nanoparticles and
[36] Y.W. Wang, L. Chen, M. Liang, H. Xu, S. Tang, H.H. Yang, et al., Sensitive
SC R
fluorescence immunoassay of alpha-fetoprotein through copper ions modulated growth of quantum dots in-situ, Sensor. Actuat. B-Chem. 247 (2017) 408-413.
[37] K. Sheng, W. Liu, L. Xu, Y. Jiang, X. Zhang, B. Dong, et al., An ultra-sensitive
U
label-free immunosensor toward alpha-fetoprotein detection based on three-
N
dimensional ordered IrOx inverse opals, Sensor. Actuat. B-Chem. 254 (2017) 660-668. [38] L. Liu, L. Tian, G. Zhao, Y. Huang, Q. Wei, W. Cao, Ultrasensitive electrochemical
A
immunosensor for alpha fetoprotein detection based on platinum nanoparticles
PT
Biographies
ED
Acta 986 (2017) 138-44.
M
anchored on cobalt oxide/graphene nanosheets for signal amplification, Anal. Chim.
Ying Shi is a doctoral student in School of Chemistry and Chemical Engineering,
CC E
Southwest University, China. Her major research interest is molecular spectrum analysis and optical biosensors construction.
A
Miao Yang received her M.S. degree in chemistry at Southwest University in 2016. Now she is a doctoral student in School of Chemistry and Chemical Engineering, Southwest University, China. Her major research interest is chemical theory and computation.
Li Liu received her Ph.D. degree in chemistry at Southwest University in 2017. Now she is a lecturer in College of Chemistry and Environmental Science at Qujing Normal University, China. Her research interests are sensors and spectrum analysis. Yanjiao Pang is master student in chemistry at Southwest University in 2016. Her major research interest is molecular spectrum analysis and biosensor construction.
IP T
Yijuan Long received her Ph.D. degree in chemistry at Southwest University in 2013. Now she is a senior experimentalist in School of Chemistry and Chemical
SC R
Engineering, Southwest University, China. Her research interests is spectrum analysis.
Huzhi Zheng received his Ph.D. degree in chemistry at Wuhan University in 2005.
U
Now he is a professor in School of Chemistry and Chemical Engineering, Southwest
ED
M
A
mimics and their application for biosensing.
N
University, China. His main research interests lie in drug delivery, novel enzyme
Figure and table captions
PT
Fig. 1. The peroxidase-like activity of GTP. (a) Schematic illustration and (b) absorption spectra of GTP-catalyzed oxidation of TMB by H2O2 to form oxTMB in
CC E
pH 5.0 PBS buffer. (c) Absorbance value changes with different concentrations of GTP-catalyzed oxidation of TMB in the presence of H2O2. Fig. 2. Production of hydroxyl radicals catalyzed by GTP. (a) The catalytic reaction in
A
the presence of different concentration of radical scavengers. (b) ESR spectra of H2O2/DMPO spin adduct produced in PBS buffer (pH 5.0) containing 50 mM DMPO, 10 mM H2O2, and various concentrations of catalyst. Fig. 3. Steady-state kinetic assays of GTP. (a) The concentration of H2O2 was 10 mM and the TMB concentration was varied. (b) The concentration of TMB was 0.80 mM
and the H2O2 concentration was varied. Error bars represent the standard deviations of three independent experiments. Fig. 4. ALP sensing based on GTP-mediated enzyme cascade reaction. (a) Principle illustration of the colorimetric sensor for ALP detection. (b) Absorption spectra of GTP-catalyzed TMB-H2O2 system in the presence of ALP with different units (0,
IP T
0.01, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 100, 150, 200 U/L). (c) Calibration curve for ALP detection. (d) The linearity of logarithmic absorbance changes with respect to
logarithmic ALP activity. (e) The selectivity of the GTP-based method for ALP assay.
SC R
A0 represents the reaction without ALP and A represents the reaction with different ALP levels.
U
Fig. 5. AFP detection based on GTP-mediated enzyme cascade reaction. (a)
Schematic illustration of the working principle for AFP. (b) Photographs and (c) the
N
corresponding absorption spectra of the ALP-linked immunoassay integrating with
A
the peroxidase-like activity of GTP in the presence of different amounts of AFP (0,
M
0.5, 1, 5, 10, 20, 30, 50, 100, 150 ng/mL). (d) Calibration curve for AFP detection. Error bars were estimated from three replicate measurements. A0 represents the
ED
reaction without AFP and A represents the reaction with different concentrations of AFP.
PT
Table1. Comparison of the apparent Michaelis-Menten constant and maximal
A
CC E
velocity of GTP, other small moleculer and HRP.
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-1
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-2
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-3
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-4
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-5
Table1. Comparison of the apparent Michaelis-Menten constant and maximal velocity of GTP, other small moleculer, and HRP.
1.0×10-5
F[29]
1.0×10-4
CF[30]
1.0×10-4
AF[30]
1.0×10-4
DCF[32]
1.0×10-4
DFF[31]
1.0×10-4
L-Glu[33]
1.0×10-2
L-Asp[33]
1.0×10-2
HRP[33]
2.3×10-11
Km (mM) 2.93±0.219 0.761±0.001 1.31±0.129 1.11±0.044 0.304±0.026 1.66 ± 0.042 1.90 ± 0.126 1.86 ± 0.232 0.21 0.06 1.78 2.97 0.6268±0.219 0.0144±0.002 0.4370±0.083 0.0115±0.001 0.179±0.020 1.18±0.147
N A M ED PT CC E A
Vmax (10-8 M s-1) 9.15±0.052 1.80±0.002 0.418 ± 0.11 0.438 ± 0.18 0.547 ± 0.10 0.836 ± 0.12 0.384 ± 0.16 0.217 ± 0.02 0.207 0.096 0.404 0.983 0.2287±0.052 0.1927±0.006 0.3560±0.040 0.2713±0.003 3.895±0.218 7.307 ± 0.824
kcat (s-1) [a] 9.15×10-3 1.80×10-3 4.18×10-5 4.38×10-5 5.47×10-5 8.36×10-5 3.84×10-5 2.17×10-5 2.07×10-5 0.96×10-5 4.04×10-5 9.83×10-5 2.29×10-7 1.93×10-7 3.56×10-7 2.71×10-7 1.69×103 3.18×103
IP T
GTP
Substrate TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2
SC R
[E] (M)
U
Catalyst