A Simple and Convenient Aptasensor for Protein Using an Electronic

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A Simple and Convenient Aptasensor for Protein Using Electronic Balance as Readout Alian Wang, Xiaoming Ma, Yanzhu Ye, Fang Luo, Longhua Guo, Bin Qiu, Zhenyu Lin, and Guonan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03823 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Analytical Chemistry

A Simple and Convenient Aptasensor for Protein Using Electronic Balance as Readout Alian Wang, a Xiaoming Ma,b Yanzhu Ye,a Fang Luo,*,b,c Longhua Guo, b Bin Qiu,b Zhenyu Lin*,b and Guonan Chenb

a

Department of Science Research and Training, Fujian Institute of Education, Fuzhou

350001, China

b

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian

Provincial Key Laboratory of Analysis and Detection for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China

c

College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian

350116, China

Corresponding author: Fang Luo, Zhenyu Lin Email: [email protected], [email protected]

Address: College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China.

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Electronic balance, one of the most common equipments in the laboratory, is normally adopted to directly measure the weight of the target with high accuracy, however little attention has been paid on the extension of its application. In this study, electronic balance was used as readout to develop a novel aptasensor for protein quantification for the first time. Thrombin was selected as a model target and its two aptamers recognizing different sites of the protein were used (one aptamer was immobilized on the surface of magnetic microparticles and the other aptamer was functionalized with platinum nanoparticles). The two aptamers were specifically bound with the thrombin to form a sandwich structure, thus the platinum nanoparticles were linked to the magnetic microparticles and they were separated by a magnet easily. The captured platinum nanoparticles effectively catalyzed the decomposition of H2O2, generating a large volume of O2 to discharge a certain amount of water in a drainage device because the pressure in vial is higher than that outside vial, the weight of water was accurately measured by an electronic balance. The weight of water increased with the increasing of the thrombin concentration in the range of 0 to 100 nM with a detection limit of 2.8 nM. It is the first time to report the use of an electronic balance as signal readout for biomolecule quantitation in bioassay.

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INTRODUCTION Measurements of signal of biosensors are typically accomplished using electrochemical1, fluorescent2, or chemiluminescent3 readout, however most of these readout methods rely on bulky and expensive analytical equipments and skilled technicians. Consequently, it is necessary to search a simple, low-cost and convenient readout technology for biosensors. Electronic balance, the most common and necessary device in the laboratory, has not yet used as the signal readout of biosensors. In the determination of trace substance, the quantity of the analyte is too low to be detected directly by an electronic balance. To employ electronic balance as the novel readout tool, signal conversion and amplification is needed. Platinum nanoparticles (PtNPs) have the excellent ability to catalyze the decomposition of H2O2 to H2O and O24. Given the significant increase in gas pressure of the closed system due to the catalyzed decomposition of H2O2 via PtNPs, the molecular recognition can be transformed into a pressure signal. On the basis of this phenomenon, several methods have been reported for biochemical targets detection. For example, pressure-based bioassays have been developed for protein detection using a hand-held pressuremeter or differential pressure gauge as readout5-8. Besides, some distance-readout methods based on the decomposition of H2O2 to O2 to move an ink bar in a volumetric bar chart chip9-10 or a capillary tube11 have been developed for directly visual quantification of biomarkers. However, a pressure meter that is not commonly used in the laboratory is required in the pressure-based bioassays, and the distance-readout methods are apparent short of accuracy and repeatability when using 3



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the ink level as an indicator. Furthermore, the sensitivity of these readout equipments is not high. More improvement is necessary for the pressured-based bioassay. Compared with the abovementioned signal readout, electronic balance is more promising as the signal readout because it is one of the most accurate analysis tools. When the electronic balance is used as the signal readout, the pressure difference caused by the continuous generation of O2 can push the water out in a drainage device, thus the pressure signal can be converted to an amplified weight signal. In this context, we developed a novel aptasensor for protein detection based on the conversion of molecular recognition to a weight signal using electronic balance as readout. Thrombin, a coagulation protein in the bloodstream12, plays a central role in a number of cardiovascular diseases13 and therefore it was used as the model target. In this strategy, the molecular recognition can be converted into a weight signal, thus allowing sensitive and quantitative detection of thrombin by electronic balance.

EXPERIMENTAL SECTION Materials. Dynabeadss™ MyOne™ Carboxylic Acid (1.05 µm, 10 mg/mL) was purchased from Invitrogen (Norway). Thrombin, L-cysteine, trypsin, bovine serum albumin

(BSA),

cytochrome,

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

ehydrochloride (EDC), 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Sigma-Aldrich (Shanghai, China). Streptavidin was obtained from Sangon Biotechnology

Co.

(Shanghai,

China).

Chloroplatinic

acid

hexahydrate

(H2PtCl6·6H2O), ascorbic acid, boric acid, sodium tetraborate, tris (hydroxymethyl) 4


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aminomethane (Tris), ethylenediaminetetraacetic acid disodium salt (EDTA), hydrogen peroxide (H2O2, 30 wt %), Tween-20 were purchased from Aladdin Chemistry Co. (Shanghai, China). The preparation of aptamer (Apt29) functionalized magnetic microparticles (Apt29-MMPs) and aptamer (Apt15) functionalized platinum nanoparticles (Apt15–PtNPs) was detailed in Supporting Information. Glass vials and plastic screw caps with silicon septa were purchased from Agilent Technologies (Santa Clara, USA). All other reagents were analytical grade and used as received. Deionized water obtained from a Millipore water purification system (Milli-Q, Millipore, resistance 18.2 MΩ.cm ) was used throughout all project. The buffers are as follows: Buffer A (binding buffer): 20 mM tris-HCl, 140 mM NaCl, 1 mM MgCl2, 5 mM KCl, 1 mM CaCl2, pH 7.4; Buffer B (washing buffer): buffer A containing 0.1% Tween-20. All oligonucleotides used in this study were ordered from Sangon Biological Co. (Shanghai, China). Their sequences are listed as follows: 15-mer aptamer (Apt15): 5'-Biotin-TTT TTT TTT TTT GGT TGG TGT GGT TGG. 29-mer aptamer (Apt29): 5'-NH2-(CH2)6TTT TTT TTT AGT CCG TGG TAG GGC AGG TTG GGG TGA CT. Apparatus. Transmission electron microscopy (TEM, Tecnai G2 F20 S-TWIN, FEI, USA) was used for the characterization of prepared streptavidin functionalized platinum nanoparticles. An electronic balance (Sartorius, BS124S, China) with 5



accuracy of 0.1 mg was used to record the weight of water.

RESULTS AND DISCUSSION Principle of Aptasensor for Thrombin Using Weight as Signal Out. Scheme 1 shows the principle of aptasensor for thrombin based on the conversion of molecular recognition to a weight signal using electronic balance as readout. Two aptamers (Apt15 and Apt29) of thrombin were selected. Apt15 and Apt29 can bind to the fibrinogen-binding site and the heparin-binding site of thrombin15,16, respectively. Apt29 was immobilized on the surface of MMPs to capture thrombin, while Apt15 was modified on the surface of PtNPs. In the presence of thrombin, a sandwich aptamer complex was formed and therefore the PtNPs was attached onto the surfaces of MMPs. This mixture was separated easily by a magnet and subsequently dispersed in H2O2, and then the PtNPs-MMPs/H2O2 mixture was immediately transferred into a drainage device. A glass vial (10 mL) sealed with a cap containing a silicon septum was divided into two parts; one part was used to place the generated gas complex (~1.5 mL) and the other part was filled with water (~7.0 mL), and a pipe communicated with the atmosphere was inserted into the bottom and the top of glass vial was interlinked. A large amount of O2 was generated in the gas generation part due to the decomposition of H2O2, resulting in an enhancement of pressure inside the glass vial. A certain amount of water overflowed the pipe with the increase of pressure, the water was collected in a container and its weight was measured by electronic balance. As the concentration of PtNPs is strictly proportional to that of thrombin, a 6


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close relationship between the weight of water and the target can be established. On the basis of this principle, a simple aptasensor for thrombin using a convenient electronic balance as readout can be designed. Furthermore, as the PtNPs cannot be poisoned in H2O2, we infer that the sensitivity may be adjustable by changing the reaction time to meet specific requirement. Catalytic Ability of the PtNPs-streptavidin Conjugation. The PtNPs-streptavidin conjugation (PtNPs-SA) was characterized by TEM, the result shows that it had good dispersion and uniform size with an average size around 50 nm (see Figure S-1 in Supporting Information). Then, the catalytic ability of PtNPs-SA for H2O2 decomposition was studied. As shown in Figure 1A, the weight of overflowed water increased almost linearly with the reaction time, indicating that the catalytic production of O2 by PtNPs-SA is a zero-order reaction11. Furthermore, there was a linear relationship between the weight of overflowed water and the concentration of PtNPs-SA at different reaction times (Figure 1B). These results indicate that the synthesized PtNPs-SA possesses excellent catalytic property toward the decomposition of H2O2.

Optimization of the Reaction Conditions. To achieve superior sensitivity, different experimental parameters such as the concentrations of Apt29-MMPs and Apt15-PtNPs and incubation time were investigated. The addition of a higher concentration of Apt29-MMPs to the assay can result in more thrombin being carried forward to PtNPs addition step and hence a strong signal is obtained. However, a 7



larger concentration of Apt29-MMPs added may lead to the increase of the thrombin-independent signal (background), causing a decreasing sensitivity. So we firstly optimized the concentration of the Apt29-MMPs (0.125, 0.25 and 0.375 mg/mL) in the assay. As shown in Figure 2A, the unspecific signal (0 nM thrombin) was very weak when the concentrations of Apt29-MMPs were 0.125 and 0.25 mg/mL, while a remarkably increasing signal was observed at 0.375 mg/mL. Considering the strong signal due to the specific binding and the unspecific signal, 0.25 mg/mL was chosen as the optimum concentration for Apt29-MMPs. Because Apt15-PtNPs are crucial for the decomposition of H2O2 to O2, the concentration of Apt15-PtNPs (0.25 ~ 3.75 nM) was also investigated. With the increase of Apt15-PtNPs, the signal toward thrombin (50 nM) and nonspecific catalytic effect of Apt15-PtNPs were increased, and a maximal difference existed between such signal and background signal when the concentration was 1.25 nM (Figure 2B). Therefore, 1.25 nM of Apt15-PtNPs was chosen in this assay. The incubation time for the interaction between Apt29-MMPs and thrombin was investigated. With the increasing of the reaction time, the overflowed water increased and a maximum value was obtained at 1.5 h (Figure 2C), so 1.5 h was adopted as the optimum for the binding of thrombin to Apt29-MMPs. Furthermore, the effect of incubation time between the thrombin-Apt29-MMPs conjugate and Apt15-PtNPs was also investigated (Figure 2D). The weight of overflowed water increased with the increasing incubation time from 10 min to 2 h and then reached a platform over 2 h, therefore 2 h was used as optimum incubation time between thrombin-Apt29-MMPs 8


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and Apt15-PtNPs. Calibration Curve and Selectivity of the Proposed Aptasensor. The pressure inside the glass vial gradually increased due to the decomposition of H2O2, which pushed out a certain amount of water, and the weight of water was accurately measured by electronic balance. Figure 3 shows that the weight of water increased gradually with the increase of the thrombin concentration (the catalytic decomposition reaction time was 15 min), and there was a good linear relationship between the weight of water and the concentration of thrombin in the range from 0 to 100 nM, the regression equation is as follows: W = 0.04766 C - 0.01919 (R2 = 0.9985) where W is the weight of water pushed out, C is the concentration of thrombin, and R2 is the correlation coefficient. The limit of detection (LOD) was 2.8 nM (defined as background value + 3×the standard deviation). Table S-1 displays the comparative LOD between our aptasensor and other reported approaches for thrombin. Although some conventional methods show a lower LOD for thrombin, such as fluorescence (~ 0.2 nM), electrochemistry (~ 5.6 pM), and chemiluminescence (~100 pM), yet they rely on professional and expensive instruments, whereas the electronic balance was used as readout in our method and it is the essential instrument but with high accuracy in the laboratory. Besides, as the PtNPs cannot be poisoned in H2O2, the sensitivity may be adjustable by changing the reaction time in this method. To verify this inference, an assay was investigated. Figure S-2 shows that when the catalytic reaction time was prolonged, the increase rate of the signal of thrombin was greater 9



than that of the background, thus the signal of noise ratio (S/N) was increased. In this way, the LOD can be further lowered by prolonging the catalytic decomposition reaction time. Intraassay coefficient of variation (CV) were calculated for each concentration of thrombin (n = 6) and the CVs corresponding to 10 nM, 25 nM, 50 nM, 75 nM, 100 nM were 9.32%, 9.08%, 6.70%, 3.43%, 7.69%, respectively. The CVs over the entire range of concentrations were below 10%, which confirms that the proposed aptasensor is reproducible. To evaluate the selectivity of this method, thrombin and other proteins such as L-cysteine, trypsin, cytochrome C, BSA were employed. The concentration of thrombin was set as 10 nM, which is ten times lower than that of other control proteins. As shown in Figure 4, water was significantly overflowed in the presence of thrombin but no remarkable changes were observed upon the addition of other proteins. These results suggest that the developed method exhibits good selectivity. The Analysis of Real Samples. The application of the proposed strategy was investigated by detecting thrombin in diluted human serum. Human serum was provided by the Mengchao Hepatobiliary Hospital (Fuzhou, China), and the serum sample was diluted by buffer A before use. Different concentrations of thrombin (10, 50, 90 nM) were spiked into 100-fold diluted human serum, and then measured by the proposed method. As shown in Table S-2, the recovery rate varied from 89.6% to 95.1%, demonstrating that the aptasensor has a promising feature for the application in real biological samples.

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CONCLUSIONS In summary, a simple and convenient aptasensor has been developed for thrombin based on converting molecular recognition to a weight signal using electronic balance as readout. This aptasensor has high sensitivity and selectivity for thrombin, and the strategy has been applied to detect thrombin in human serum with good result. As the electronic balance is one essential instrument with high accuracy, the method has great potential for developing a simple and highly sensitive biosensing platform. Moreover, given that many platforms are realized via immunization, so this method may combine with the immunoassay technique to achieve a promising platform for ultrasensitive bioanalysis for different targets. ACKNOWLEDGMENTS This project was partly financially supported by National Sciences Foundation of China (21575025, 21575027), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), the Foundation of Fujian Educational Committee (JA15611) and the Foundation for Scholars of Fuzhou University (XRC-1671, XRC-17007).

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Figures and Captions Scheme 1. Principle of aptasensor for thrombin using electronic balance as signal readout.

Figure 1. (A) Time-dependent changes in weight of water with different concentration of PtNPs-SA in H2O2 (0.5 mL, 30%) at different reaction times: (a) 0.025 nM, (b) 0.05 nM, (c) 0.1 nM, (d) 0.15 nM, (e) 0.2 nM. (B) Linear relationship between the weight of water and the concentration of PtNPs-SA at different decomposition reaction time, 15 min (a) and 30 min (b). Error bars represent the standard deviations for three replicates.

Figure 2. Effects of (A) the amount of Apt29-MMPs, (B) the concentration of Apt15-PtNPs, (C) the incubation time for the interaction between Apt29-MMPs and thrombin, (D) the incubation time between the thrombin-Apt29-MMPs conjugate and Apt15-PtNPs. The 50 nM thrombin was used to optimize the incubation time. Error bars represent the standard deviations for three replicates.

Figure 3. Liner relationship between the weight of water and thrombin concentration. Error bars represent the standard deviations for three replicates.

Figure 4. Specificity of the assay for thrombin detection with different proteins. Error bars represent the standard deviations for three replicates. 14


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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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A Simple and Convenient Aptasensor for Protein Using an Electronic

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