Paraoxon, non-recombinant human AChE from red blood cells, acetylthiocholine iodide, zirconium oxychloride (ZrOCl2), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), hydroxylamine, CNBr, alpha-chymotrypsin (type I–S), 2-mercaptoethanol, N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES) buffer, horseradish peroxidase (HRP) and 5,5-dithio-bis(2-nitrobenzoic acid) were purchased from Sigma-Aldrich. Polyclonal and monoclonal anti-AChE antibodies were purchased from Abcam. A Qdot@655 antibody conjugation kit was purchased from Invitrogen. Human plasma was purchased from Golden West Biologicals, Inc.. Other reagents were commercially available and were of analytical reagent grade. Solutions were prepared with ultrapure (18MΩ) water from a Millipore Milli-Q water purification system (Billerica, MA).
Square-wave voltammetric (SWV) measurements were performed using an electrochemical analyzer CHI 660 (CH Instruments, Austin, TX) connected to a personal computer. A disposable screen-printed electrode (SPE) consisting of a carbon working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode was purchased from Alderon Biosciences, Inc. for electrochemical measurements. A sensor connector (Alderon Biosciences, Inc.) allows for connecting the SPE to the CHI electrochemical analyzer. LC MS/MS analyses of the phosphorylated AChE was performed using the Agilent 1200 series LC system, Agilent HPLC-Chip cube and Agilent 6330 XCT ion trap. Centrifugal filter devices (AmiconUltra-15, 30000 MWCO, Millipore Corp.) were used to separate and concentrate the sample solution. Dialysis was performed with Float-A-Lyzer (MWCO 25 000, Spectrum Laboratories, Inc.). The Disposable PD-10 desalting column packed with Sephadex G-25 medium (exclusion limit 5000) was purchased from Amersham Bioscience Corp. and used to purify the protein solution. Centrifugation was performed with a Sorvall RC 26 plus (Kendro Laboratory Product).
Preparation of phosphorylated AChE adducts
AChE was diluted in 0.01-M phosphate buffer (pH 7.4) (final concentration, 110 nM) before paraoxon addition. Paraoxon was diluted in an appropriate vehicle, such as acetone, at less than 5% of the total volume and had no significant effect on AChE activity or reactivity with anti-AChE. The final concentration of paraoxon was 750 µM. AChE was incubated with paraoxon for 2 h at room temperature. The decrease in enzyme activity was monitored until inhibition was complete. The solution was dialyzed against 0.01 M phosphate buffer with saline (PBS) (2×1 L) overnight at 4°C, to remove the excess of paraoxon. Finally, a volume of phosphorylated AChE was decreased to approximately 0.4 mL by ultrafiltration (Millipore Ultrafree-MC, Bedford, MA). The protein content of the concentration was determined spectrophotometrically at 280 nm using an absorption coefficients of E1%= 16.
Preparation of phosphorylated AChE samples for mass spectrometry (MS) analysis
Prior to the MS characterization, phosphorylated AChE was precipitated with ice-cold acetone (1:4 vol/vol), the acetone was decanted, and the remaining pellet dried in a speed-vac. The pellet was resolubilized in 100 µL of degassed 0.1N HCl and 2.5 µL of 20 nmol/µL CNBr was added (a 20-fold excess) to chemically cleave the lipophilic protein at the C-terminus of methionine residues for 12 h at room temperature, in the dark. CNBr was removed by adding 200 µL of acetonitrile and lyophilizing the sample to dryness in a speed-vac. Finally, the pellet was resolubilized in 200 µL of 50 mM ammonium bicarbonate, pH 7.8 and treated with 10 µL of 0.1 µg/µL chymotrypsin at a protein to enzyme ratio of 1:20 for 12h, at 37°C. Proteolysis was stopped by flash freezing at −80 °C.
Mass spectrometry analyses
LC MS/MS analyses of the huAChE peptides was performed using the Agilent 1200 series LC system, Agilent HPLC-Chip cube and Agilent 6330 XCT ion trap. 100 nL of peptide solution were loaded onto a 4 mm, 40 nL enrichment column and separated on a polymer embedded 75 µm × 43 mm column containing 5 µm, 300 A° ZORBAX SB-C18. The mobile phases employed were: 0.1% formic acid in water (A phase) and 90% acetonitrile with 0.1% formic acid (B phase). After initial loading at 3% B, the gradient increased to 70% B over 10 minutes then ramped to 95% B in two minutes. The analytical flow rate was 600 nL/min. Peptides were ionized using nano-electrospray in the positive mode and automatically detected in MS/MS mode from 50 – 2200 m/z. Peptide mass mapping of MS/MS data was performed using MASCOT® (MatrixScience) after configuring the “enzyme file” to include a CNBr/Chymotrypsin cleavage and configuring the “modifications file” to include the following variable modifications: homoserine and homoserine lactone at C-terminal methionines, diethylphosphorylation (DEP) of serine with neutral scan loss of 154.0395 (monoisotopic), and monoethylphosphorylation of serine (which results from “aging” of the DEP adduct) with a neutral scan loss of 126.0082 (monoisotopic).
AChE and phosphorylated AChE activity assay
A modified Ellman assay was performed essentially as described. [20
] AChE or phosphorylated AChE was diluted serially in 0.1-M phosphate buffer (pH 7.4) and 2 µL added to a microtiter plate. The plate substrate acetylthiocholine iodide (ATCh-I; 2 µL) provided a final concentration of 0.75 mM. A 5, 5-dithio-bis (2-nitrobenzoic acid) (DNTB) was added to the assay solution to give a final concentration of 0.32 mM. Absorbance was measured at OD 412 nm over a 3-min period at 25°C on a versaMax microplate reader with SOFTMAX PRO v. 3.0 software (Molecular Devices).
Fourier Transform Infrared Spectroscopic Characterization of phosphorylated AChE adducts
Infrared spectra were collected using a Bruker IFS66/S Fourier Transform Infrared Spectrometer (FTIR) with a Michelson interferometer. The spectrometer was purged for 10 minutes with nitrogen gas before spectral collection to decrease strong absorbances from atmospheric carbon dioxide and water vapor. Spectra were collected using a mid-infrared Globar source, a KBr beamsplitter, and a deuterated triglycine sulfate (DTGS) detector. Spectra consisted of 512 co-added scans acquired using double-sided/forward-backward mirror motion with a 10 kHz mirror velocity. Each spectrum took over seven minutes to acquire. Spectra were collected at 4 cm−1 resolution over the frequency range of 5200 to 500 cm−1, with a 16-cm−1 phase resolution and a zero filling factor of 2, using a Blackman Harris three-term apodization, and Mertz phase correction. A 16-kHz low-pass filter was used to prevent aliasing. Absorbance spectra were obtained by rationing the attenuated total reflectance (ATR) spectra of the samples to the ATR spectra of the uncoated diamond crystal.
Preparation of HRP- anti-AChE conjugate
The HRP-anti-AChE conjugate was prepared by following a modified procedure described by Graback and Gergely.[40
] Briefly, HRP was activated by adding 0.5 mg HRP in 1 mL of activation buffer containing 0.1-M MES and 0.5-M NaCl (pH 6.0). A total of 0.4 mg of EDC (final concentration ~2 mM) and 0.6 mg of NHS (final concentration ~5 mM) were added to the activated HRP solution, and reacted for 15 minutes at room temperature. The EDC was quenched by adding 1.4 µL of 2-mercaptoethanol (final concentration of 20 mM). The excess reducing agent and inactivated crosslinker were removed with a PD-10 column. The eluent was collected and concentrated to 1 mL in an ultracentrifuge tube. An amount of Anti-AChE was added at an equal molar equivalent to the HRP in the above solution, and the reaction proceeded for 2 hours at room temperature. The reaction was quenched by adding hydroxylamine to a final concentration of 10 mM. Excess hydroxylamine was removed using a PD-10 column and the eluent was collected and kept at 4°C for further use.
Preparation of QD-anti-AChE antibody conjugates
Qdot@655 antibody conjugation kit from Invitrogen was used to conjugate the anti-hAChE antibody to QDs. Before conjugation, anti-AChE from a human was purified with a gel-filtration column (Superose 12, Pharmacia-LKB) to remove surfactants and other proteins, such as bovine serum albumin (BSA). The collected eluent fractions (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4) were mixed and concentrated to 0.3 mL by a centrifugal filter device (Amicon Ultra-15). The concentration of anti-AChE was ~ 0.5 mg/mL. Conjugation was performed according to the manufacturer’s procedure. Anti-AChE was reduced with dithiothreitol (DTT) to expose free sulfhydryls and excess DTT was removed by size-exclusion chromatography. QDs were activated using the hetero-bifunctional crosslinker, 4-(maleimidomethyl-1cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), yielding a maleimide-nanocrystal surface. Excess SMCC was removed by size-exclusion chromatography. Activated QDs were mixed with reduced anti-AChE and a coupling reaction proceeded for 1 hr. The reaction was quenched by adding β-mercaptoethanol. The produced conjugate was concentrated by ultrafiltration and purified using size-exclusion chromatography. The eluted QDs-anti-AChE conjugate was collected and then stored at 4°C for further use.
Preparation of ZrO2 nanoparticle-coated SPE
Before experiments, the screen printed electrode (SPE) was washed with distilled water and dried with nitrogen. ZrO2
nanoparticles were deposited onto bare SPE in an aqueous electrolyte of 5.0 mM ZrOCl2
and 0.1-M KCl by cycling the potential between −1.1 and + 0.7 V (versus Ag/AgCl) at a scan rate of 20 mV/s for 10 consecutive scans.36
The SPE modified with zirconia nanoparticles (ZrO2
/SPE) was rinsed with water and dried with N2
prior to use.
Enzyme linked immunosorbent assay of phosphorylated AChE adducts
Fifty microliters of phosphorylated AChE adduct or purified AChE with the desired concentration was placed in each microtiter plate well. Control experiments were performed by adding 50 µL of blocking buffer (PBS containing 1% BSA) in the microplate wells. The plate was covered with an adhesive plastic and incubated overnight at 4°C. The coating solution was then removed, and the plate washed twice by filling the wells with 200 µL PBS. The washes were removed by flicking the plate over a sink. The remaining drops were removed by patting the plate on a paper towel. The remaining protein-binding sites in the coated wells were blocked by adding 200 µL of blocking buffer per well. The plate was incubated for 2 h at room temperature. The plate was then washed twice with PBS. One-hundred milliliters of HRP-Anti-AChE (1/100) in blocking solution was added and the immunoreaction proceeded for 2 h at room temperature. The plate was then washed four times with PBS. One-hundred microliters of substrate (TMB+H2O2) was added to each well and the enzyme catalyzed reaction progressed for 20 minutes at room temperature. One-hundred microliters of 1 M hydrochloric acid was then added to stop the enzymatic reaction, and the plate was read on a microplate reader at 450 nm.
Electrochemical immunosensing phosphorylated AChE with ZrO2/SPE and QD labeled anti-AChE
Fifty microliters of phosphorylated AChE solution at the desired concentration was aliquoted atop a ZrO2-coated SPE surface and incubated for 1 hour in a humidified chamber. The phosphorylated AChE attached SPE was then washed intensively with washing buffer (0.05-M phosphate buffer containing 0.1% w/w Tween, pH 7.4). The washing included a 1-min rinse from a washing bottle followed by a 5-min wash in a Millipore stirring cell (model 8010). The SPE was blocked for 30 min with 0.5% BSA in 0.05-M phosphate buffer. After washing, the SPE was incubated with 10 µL of QD-anti-AChE(1/50, v/v) for 1 hr in the humidified chamber, then thoroughly rinsed with PBS, and then shaken with washing buffer following the above procedure to remove the physical adsorption of QD-anti-AChE on the electrode surface. The SPE was rinsed with PBS again and dried under an N2 stream. A 10-µL aliquot of 1 M HCl was dropped on the working electrode surface of the SPE to dissolve the captured QDs. Fifty microliters of detection solution containing 10 µg mL−1 Hg in 0.2-M acetate buffer (pH 4.6) was then added for electrochemical measurement. Square-wave voltammetry (SWV) measurements were performed using an in situ-plated Hg film on the SPE by a 2-min accumulation at −1.4 V. Subsequent stripping was performed after a 2-second rest period from −1.0 V to −0.5 V with a step potential of 4 mV, amplitude of 25 mV, and frequency of 15 Hz.
Organophosphates (paraoxon), cadmium, and mercury are highly toxic and the wastes containing these compounds should be collected in a specific container. Skin and eye contact and accidental inhalation or ingestion should be avoided.