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An enzyme-free amplification strategy for biosensing using Fe3+- polyglutamic acid coordination chemistry Jing Wu, Yunlei Xianyu, Xiangfeng Wang, Dehua Hu, Zhitao Zhao, Ning Lu, Meng-Xia Xie, Hongtao Lei, and Yiping Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05344 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018
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Analytical Chemistry
An enzyme-free amplification strategy for biosensing using Fe3+- polyglutamic acid coordination chemistry Jing Wua#, Yunlei Xianyuc#, Xiangfeng Wanga#, Dehua Hua, Zhitao Zhaoa, Ning Lub, Mengxia Xiea*,Hongtao Leib*, Yiping Chenc* a
Analytical & Testing Center of Beijing Normal University, Beijing 100875, China
b
Guangdong Provincial Key Laboratory of Food Quality and Safety/College of Food Science, South China Agricultural University, Guangzhou 510642, China
c
CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China
E-mail:
[email protected] (YP Chen), Phone number: (86)10 82545631 E-mail:
[email protected] (MX Xie), Phone number: (86)10 58807981 E-mail:
[email protected] (HT Lei), Phone number: (86)20 85283448
ABSTRACT: In this work, we outline a signal amplification strategy using the coordination chemistry between Fe3+ and polyglutamic acid (PGA) for biosensing applications. The theoretical calculation based on density functional theory shows that PGA has a much higher binding affinity with Fe3+ than the other metal ions. Guided by this rationale, we prepare a PGA-mediated signal probe through conjugating PGA onto polystyrene (PS) nanoparticles to form a brush-like nanostructure for Fe3+ coordination. This PGA-PS brush (PPB) has a large loading capacity of Fe3+ with a number of 1.92×108 Fe atoms per nanoparticle that greatly amplifies the signals for assays in an enzyme-free way. Combined with ferrozine coloration-based readout, this PPB-mediated amplification is further applied for the enzyme-free immunoassay that shows an ultrahigh sensitivity for detection of microcystins-LR (12 pg/mL), a 5-fold enhancement compared with that of traditional enzyme-based immunoassay (ELISA) (60 pg/mL). In addition, the good stability, rapid response and long shelf life make this enzyme-free amplification strategy a promising platform for point-ofcare biosensing applications.
Current biosensors mainly rely on polymerase chain reaction (PCR)1-6 and enzyme-catalysed reaction7-11 for signal amplification. Although they can amplify the molecular event such as the nucleic acid hybridization and antibody-antigen interaction in an exponential way, both techniques need enzymes for the assay development. In addition, the shelf life of enzymes remains an issue because they are vulnerable to the ambient conditions which impair their value in applications like on-site detections. To develop effective signal amplification strategies without aid of enzymes is urgently needed since enzyme-free amplification technique enables biosensors with rapid response and long-term stability that are highly desired for point-of-care applications. The burgeoning nanotechnology has advanced the development of enzyme-free biosensors. Different species of nanomaterials such as platinum nanoparticles12-14 and gold nanoclusters15-18 have enzyme-mimicking properties that can be employed for enzyme-free assays. However, these enzyme mimics are less efficient than the natural enzymes in most cases and their activities are easily impaired in complicated
environment that impede their real-world applications. Enzymefree biosensors have been also designed by using liposomes which are able to encapsulate signal probes for the amplification19,20. In liposome-based biosensors, the loaded molecules should be large enough otherwise the leakage issue would occur. Besides, the detergent is always needed for the release of the molecules. Compared with enzyme mimics and liposome-based encapsulation, coordination chemistry on the nanostructures provides a feasible way for the probe loading and enzyme-free means for signal amplification21-24. The coordination chemistry between metal ions and their chelators is primarily used for removing metals from industrial effluents due to their high capacities of absorption25. The chelators include activated carbon26, ethylenediamine tetraacetic acid (EDTA)27, nitrilotriacetic acid (NTA)28, and polymers such as polyacrylic acid29 and polyamino acid30. In several previous studies, poly-glutamic acid (PGA, an anionic polypeptide31) show great potential for the removal and recovery of heavy metals from industrial wastewaters due to its ability to bind several metal ions (Ni2+, Cu2+, Mn2+, Fe3+ and
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Scheme 1 Enzyme-free amplification for immunoassay using Fe3+-PGA coordination chemistry. (A) The simulated structure of PGA-metal ion complex. (B) The enzyme-free signal amplification based on the chelation between PPB and Fe3+. (C) The PPBI combined with magnetic separation and the ferrozine coloration-mediated signal readout for detection of MC-LR.
Al3+)32-35. Due to the large number of functional groups on polymers, they show a higher capacity for absorption of metal ions than small molecules. Besides the application of PGA for sewage treatment, many studies have expanded its potential for enzyme immobilization36-38 and drug delivery39 based on the high absorption capacity of PGA for various compounds. Inspired by the high binding affinity between PGA and metal ions, we outline a coordination chemistry-based strategy for loading of metal ions that acts as an enzyme-free mean for signal amplification of biosensing. Herein, we report an enzyme-free amplification strategy for biosensing using the coordination chemistry between Fe3+ and PGA (Scheme 1). The carboxyl groups in the PGA contribute to the negative charge that enables the absorption of metal ions through the electrostatic interaction (Scheme 1A). The enzymefree amplification is achieved through the PGA-mediated signal probe, which is prepared by conjugating PGA onto polystyrene nanoparticles to form a brush-like nanostructure for Fe3+ coordination (Scheme 1B). The PGA-PS brush (PPB) is expected to possess a high loading capacity of Fe3+ that allows for the signal amplification in an enzyme-free way. For proof of concept, this enzyme-free amplification strategy is applied for the PPB-mediated immunoassay (PPBI) of microcystins-LR (MC-LR), a small molecule that is the most toxic microcystin40, 41 (Scheme 1C). Antigen (bovine serum albumin-MC-LR, BSA-
MC-LR) is conjugated on PPB through EDC/NHS coupling to prepare the immuno-probe (PPB-Ag), and the capture antibody is conjugated to magnetic beads to prepare MBs-Ab. In the competitive immunoassay, PPB-Ag and MC-LR competitively react with MBs-Ab and form the immune-complex containing MBs-Ab-PPB-Ag and MBs-Ab-MC-LR. The coordination chemistry between Fe3+ and PGA is applied to chelate Fe3+ through the immune-complex. Ferrozine is further used to chelate Fe2+ (reduced from the residual Fe3+ in the solution) and generate purple-colored product that has an absorption at 562 nm for readout21. Combined with ferrozine coloration-based signal readout, this PPB-mediated probe enables the enzymefree immunoassay through the change of A562 value that originates from the different concentrations of Fe3+ in the solution. The change of A562 value (∆A562=A1-A0) can be therefore employed for the quantification of MC-LR in this competitive immunoassays.
EXPERIMENTAL SECTION Materials and Equipment Anti-microcystins-LR (MC-LR) antibody (Ab, 4.5 mg/mL) and bovine serum albumin-MC-LR antigen(Ag, 2.0 mg/mL) were made in our laboratory.
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Analytical Chemistry
1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), sulfo-N-hydroxysulfosuccinimide (S-NHS), 2-(Nmorpholino) ethanesulfonic acid (MES), ferrozine, MC-LR (1.0 mg/mL) and other analogues (MC-WR, MC-LW, MC-LY, MCRR, MC-YR, MC-LA and MC-LF) were provided by SigmaAldrich (USA). Goat anti rabbit IgG-horseradish peroxidase (HRP) and bovine serum albumin (BSA) were from Solarbio (Beijing, China). Tablets of phosphates used to prepare 0.01 M phosphate buffered saline (PBS, pH 7.4) and Tween-20 were from Amresco (USA). γ-polyglutamic acid (PGA, molecular weights of 100000 and 700000) was from Yuanye Biotechnology (Shanghai, China). Carboxyl groupfunctionalized magnetic beads (MBs, 1000 nm in size, solid content: 10 mg/mL) were purchased from Merck-Millipore (USA). Amine group-functionalized polystyrene nanoparticles (PS, 1000 nm in size, solid content: 100 mg/mL) were from Micromod (Germany). 3, 3', 5, 5'-tetramethylbenzidine (TMB) was from Beyotime (Shanghai, China). Phenanthrolin, potassium ferricyanide, ferric chloride, hydroxylamine hydrochloride, and ammonium acetate were from Beijing Chemical Works (Beijing, China). The Costar® 96-well plate was supplied by Corning Inc. (USA). The water used in the experiments was deionized by water purification system from Millipore (USA). MS-3 oscillator was purchased from IKA (Germany) to mix the reaction solution. SuperMag TM magnetic separator was purchased from Ocean NanoTech Co. Ltd. (USA) to separate the MBs. The scanning electron microscope (SEM) from Hitachi High-Technologies Corporation (Japan) was used to characterize the PS and PPB nanoparticles. The sizes and zeta potentials of PS, PPB and PPB-Fe3+ were characterized by Zetasizer Nano (NZS) purchased from Malvern (UK). The inductively coupled plasma mass spectrometry (ICP/MS) obtained from Shimadzu (Japan) was used to test the concentrations of the ions (Na+, Cu2+, Zn2+, Fe3+, Cl-, SO42- and NO3-). An automatic microplate reader from Thermo-Fisher Electron Corporation (USA) was used to measure the absorbance of the solution. Microscale thermophoresis (MST) obtained from Nano temper technologies (Germany) was used to measure the dissociation constants (Kd) of PGA for metal ions.
solution with different metal ion solutions in 1:1 of volume ratio. We further use capillaries to draw the prepared solutions of PGA, metal ions and the complex of PGA-metal ions, respectively. Finally, we use MST to test the dissociation constants (Kd) of complex of PGA-metal ions to characterize the interaction between the PGA and different metal ions. EDS-SEM analysis for PPB-Fe3+ After the chelation of PPB with Fe3+, we use the deionized water to wash the PPB-Fe3+ complex for 3 times, followed by diluting the samples in deionized water. We drop the sample on the surface of aluminum foil and let it dry at room temperature. The preparations of PS and PPB are the same as that of PPBFe3+. We also quantified the loaded amount of Fe atom per PPB nanoparticle (Equation 1), the loaded amount of PGA per PPB nanoparticle (Equation 2) and the loaded amount of free carboxyl groups per PPB nanoparticle (Equation 3). (1) A is the loaded amount of Fe atom per PPB nanoparticle; NA is the Avogadro constant, 6.02×1023; a is the weight percentage of the Fe element in the complex of PPB-Fe3+ [0.034 mg per mg of PPB-Fe3+, which was analyzed by energy dispersive spectrumSEM, (EDS-SEM)]; MFe is the molar mass of Fe; n is the number of PPB nanoparticles per mg (1.9×109).
(2) APGA is the loaded amount of PGA per PPB nanoparticle; A the loaded amount of Fe atom per PPB nanoparticle (1.92×108); MGA is the molar mass of glutamic acid; MPGA is the molar mass of PGA, 0.12 is the atom ratio of Fe/O (O atoms in the free carboxyl groups).
(3) ACOOH is the loaded amount of free carboxyl groups per PPB nanoparticle.
Preparation of PPB-BSA-MC-LR antigen (PPB-Ag) We synthesized PPB-Ag as follows: 100 mg of PGA reacted with 200 µL of EDC (10 mg/mL) and 100 µL of S-NHS (10 mg/mL) in 2-(N-morpholino) ethanesulfonic acid buffer (MES, pH=6.0, 0.01 M) for 30 min at room temperature (RT). We then added 50 mg of amine group-modified PS nanoparticles and 2 mg of Ag into the above solution to react with the activated PGA for 1 h. After removing the excess PGA and Ag by centrifuging at 6000 rpm for 10 min, we added 1 mL of PBST (PBS with 0.05% Tween) to wash the prepared PPB-Ag for three times. Finally, we dispersed the prepared PPB-Ag in 1 mL of PBS solution and stored it at 4 °C for further use.
Preparation of MBs-antibody (MBs-Ab) We synthesized MBs-Ab as follows: 5 mg of MBs reacted with 80 µL of EDC (10 mg/mL) and 40 µL of S-NHS (10 mg/mL) in 3 mL of MES buffer for 30 min at RT. After removing the extra EDC and S-NHS using a magnetic separator, we introduced 20 µL of Ab (4.5 mg/mL) into 1 mL of MBs solution (PBS solution, 0.01 M) and incubated for 1 h at RT. After removing the extra Ab and washing the complex with PBST for three times, we dispersed the MBs-Ab complex in 1 mL of PBS solution and stored at 4 °C before the next step. The process of PPBI for analysis of MC-LR
MST analysis for PPB with different metal ions
We mixed 100 µL of MBs-Ab and 1000 µL of sample and gently shook the mixture in a vortex oscillator for 5 min at room temperature. We washed the target-MBs-Ab conjugate for
We prepare 10 µM of PGA and 5 mM of Na2SO4, ZnNO3, CuCl2, and FeCl3 using deionized water. We then mix the PGA
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kcal/mol)
