Feb 7, 2018 - Herein we directly integrate receptive aptamers into a redox charging peptide ... selectivity but also that the peptide-supported aptamer film.
Letter Cite This: Anal. Chem. 2018, 90, 3005−3008
pubs.acs.org/ac
Redox Capacitive Assaying of C‑Reactive Protein at a Peptide Supported Aptamer Interface Julia Piccoli,† Robert Hein,‡ Afaf H. El-Sagheer,‡,§ Tom Brown,‡ Eduardo M. Cilli,† Paulo R. Bueno,*,† and Jason J. Davis*,‡ †
Institute of Chemistry, São Paulo State University (UNESP), 14800-900, Araraquara, São Paulo, Brazil Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, U.K. § Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, Egypt ‡
S Supporting Information *
ABSTRACT: Electrochemical immunosensors offer much in the potential translation of a lab based sensing capability to a useful “real world” platform. In previous work we have introduced an impedance-derived electrochemical capacitance spectroscopic analysis as supportive of a reagentless means of reporting on analyte target capture at suitably prepared mixed-component redox-active, antibody-modified interfaces. Herein we directly integrate receptive aptamers into a redox charging peptide support in enabling a label-free low picomolar analytical assay for C-reactive protein with a sensitivity that significantly exceeds that attainable with an analogous antibody interface.
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n recent years, there has been a growing interest in the development of optimized interfaces capable of supporting the detection of clinically relevant analytes,1 especially those translatable to rapid low cost analyses.2−4 Derived immunosensors1,5−7 (which translate a biological biomarker recognition into a measurable signal) have been based on a variety of electrochemical techniques, such as voltammetry,8 amperometry, 8,9 and electrochemical impedance spectroscopy (EIS).10−12 EIS is a natively spectroscopic and highly sensitive method13 within which interfacial charge transfer resistance Rct is most commonly assessed as a reporter of analyte recognition.14,15 Recently we introduced electrochemical impedance-derived capacitive spectroscopy13,16−18 as a labelfree, reagentless method of mapping the change in interfacial redox capacitance (Cr) as a transducer, omitting the need for a solution phase redox-probe.14,19−21 The partition of mobile electrons between an electrode and a surface tethered chargeable (redox or quantum) species generates a spectroscopically resolvable capacitance. We have shown that this interfacial signal is fundamentally quantum mechanical in nature22−25 and that the charging signature is highly sensitive to changes in local environment.18,21,23 When incorporated into a redox addressable molecular film capable of selectively binding an analyte, Cr can (where the binding site is in close proximity) become a sensitive function of target concentration. To date, such interfaces have been created by covalent immobilization of bioreceptors within a mixed self-assembled monolayer (SAM), one film component being redox active, the second serving as a receptor anchor.16,19,21 © 2018 American Chemical Society
In this work, a surface assembling redox-tagged peptide is electrode-confined and subsequently modified with an aptamer. Single-stranded DNA or RNA aptamers5,26−33 are capable of supporting a diverse array of assays with some notable advantages over antibodies.34−38 Herein we demonstrate, not only that the so-generated interface exhibits high target selectivity but also that the peptide-supported aptamer film enables notably higher assay sensitivity than achievable with an analogous antibody interface.
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EXPERIMENTAL SECTION Chemical Reagents. The synthetic CRP aptamer (5′-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT GTT TTT TTT TTT T-NH2 3′) was synthesized by standard methods (see the Supporting Information). Ultrapure water was obtained from a Milli-Q MΩ × cm system and was used in all solutions. Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH; Fmoc-Glu(OtBu)-OH, Rink Amide resin, and 3-ferrocenylpropionic anhydride were purchased from AAPPTEC and Sigma-Aldrich Co. (USA). All solvents and chemicals used were of analytical grade. Electrochemical Apparatus. An Autolab potentiostat equipped with a FRA32 module (METROHM Instruments) was used for electrochemical measurements. A three-electrode Received: December 22, 2017 Accepted: February 7, 2018 Published: February 7, 2018 3005
DOI: 10.1021/acs.analchem.7b05374 Anal. Chem. 2018, 90, 3005−3008
Letter
Analytical Chemistry
Figure 1. (a) Representative schematic of the redox charging peptide-aptamer SAM and associated voltammetric response. Dashed lines represent the potentials sampled for nonfaradaic (0.1 V) and faradaic (0.360 V) output. CVs were obtained in 20 mM TBAClO4 in ACN/H2O 1:4, at a scan rate of 0.1 V s−1. (b) Capacitive Nyquist plots of the molecular film showing its high responsiveness to CRP.
was used to calculate the real electroactive area of the electrode (subsequently used in normalization). Sensor Construction. The sensor was prepared by immersion of the clean Au electrodes in a solution containing 2 mM of redox-tagged peptide in H2O/ACN (1:1) for 16 h (25 °C). After being thoroughly rinsed with deionized water, the carboxyl groups of the glutamic acid side chain were activated with an aqueous solution of 0.4 M N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC)/0.1 M N-hydroxysuccinimide (NHS) for 30 min. The modified electrodes were then incubated in 1 μM CRP aptamer in PBS (pH 7.4) for 1 h at room temperature, afterward CV and EIS were performed to confirm immobilization. Finally an incubation in 0.1% BSA (bovine serum albumin) solution for 30 min to deactivate any remaining active carboxyl groups was performed. Calibration curves were obtained by immersion in PBS (pH 7.4) containing increasing specific quantities of CRP ranging from 10 pM to 5 nM. Incubation times were 30 min for each concentration and analysis performed after gently rinsing electrodes with PBS solution. The relative response at any specific CRP concentration was defined as
setup comprising a 2.0 mm diameter gold working electrode from METROHM, a platinum mesh counter electrode, and a silver/silver chloride (Ag|AgCl, filled with 3.0 M KCl) reference electrode was used throughout. Synthesis of Redox-Tagged Peptide. The peptide FcGlu-Ala-Ala-Cys was manually produced by solid phase peptide synthesis (SPPS) using Fmoc protocols on Rink Amide Resin (0.48 mmol g−1). Coupling was performed at a 2-fold molar excess relative to the amino component in the resin, using diisopropylcarbodiimide (Dic)/1-hydroxybenzotriazole (HOBt). Fmoc groups were deprotected using 20% 4methylpiperidine/dimethylformamide (DMF) for 80 min. The ferrocene redox probe was introduced at the N-terminus by reaction with one molar equivalent 3-ferrocenylpropionic anhydride in 5 mL DCM/DMF (1:1) for 24 h. Peptide cleavage from the resin and removal of the side chain protecting groups were performed with 94% trifluoroacetic acid (TFA), 2.5% 1,2-ethanedithiol, 2.5% H2O and 1% triisopropylsilane for 2 h. The peptide was then precipitated with diethyl ether and separated from soluble nonpeptide material by centrifugation. The residue was extracted in a 1:1 mixture of solvent A (0.045% (v/v) TFA/H2O) and solvent B (0.036% (v/v) TFA/ acetonitrile (ACN)) and lyophilized. The crude product was purified by HPLC on a Beckman System Gold using a semipreparative reverse phase Phenomenex Jupiter C18 column (250 mm × 10 mm), packed with spherical 5 μm particles and 300 Å pore size. A linear gradient elution was employed from 20 to 50% of solvent B for 90 min. The flow rate was 5 mL min−1 at room temperature, and the injection volume 5 mL with UV detection at 220 nm. The purity of peptide was confirmed (Figure S1) using an analytical Shimadzu system with a reverse phase Phenomenex Jupiter C18 column (150 mm × 4.6 mm), packed with spherical 5 μm particles and 300 Å pore size, using a linear gradient of 5−95% of solvent B for 30 min, a flow rate of 1.0 mL min−1, and UV detection at 220 nm. The peptide was analyzed (635 g/mol) in an ion-trap mass spectrometer (Bruker) in positive mode (Figure S1). Electrode Pretreatment. Gold electrode surfaces were cleaned by mechanical polishing with aluminum oxide pads with particle of size of 0.05 μm prior to electrochemically polishing in 0.5 M NaOH between −0.7 V and −1.7 V (500 cycles). Electrodes were then immersed in EtOH under stirring for 20 min. A series of wider range scans, from −0.1 to 1.4 V, were then conducted in 0.5 M H2SO4 at a scan rate of 0.1 V s−1. The reduction peak of the gold oxide layer formed anodically
RR%CRP = [(RR CRP − RR Blank) ÷ RR Blank)] × 100
(1)
where RRCRP is the inverse of redox capacitance (1/Cr) at a specific concentration of CRP. To evaluate interfacial specificity, surfaces were incubated in HSA (human serum albumin; 1 mM) solution for 30 min prior to redox capacitance analysis. Electrochemical Measurements. All electrochemical measurements were carried out in a cell containing 20 mM tetrabutylammonium perchlorate (TBAClO4) supporting electrolyte dissolved in acetonitrile and water (1:4 (v/v)). CV was performed at a scan rate of 100 mV s−1 between 0.0 and 0.7 V. Electrochemical impedance measurements were carried out in the ac frequency range of 100 kHz to 0.1 Hz with 10 mV amplitude (peak to peak). The dc bias potential was set to the formal potential of the ferrocene redox-tagged peptide (0.36 V, determined by CV). Measurements were verified for compliance with Kramers−Kronig linear system theory. Impedancederived capacitance analyses utilized the relationship C*(ω) = 1/iωZ*(ω), where ω is the angular frequency and i is the complex number = −1 .14 3006
DOI: 10.1021/acs.analchem.7b05374 Anal. Chem. 2018, 90, 3005−3008
Letter
Analytical Chemistry
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RESULTS AND DISCUSSION The interface utilized in the present work comprises a simple electrochemically active peptide sequence (Fc-Glu-Ala-Ala-Cys, see Figure S2) obtained through low cost solid phase peptide synthesis (SPPS39). Synthesis commenced from the C-terminal cysteine, used for electrode surface anchoring.40 Two alanine residues were introduced to promote film crystallinity,40 then an N-terminal glutamic acid integrated. A ferrocene redoxprobe was introduced at the N-terminus by reaction with 3ferrocenylpropionic anhydride with the carboxyl side-chain remaining unmodified to enable subsequent bioconjugation41 to the SAM. Upon cleavage of the peptide from the Rink Amide resin, the C-terminal group remained amidated to avoid negative charges which could impede lateral packing of peptide chains by electrostatic repulsion.40 The structure and purity of the peptide was confirmed by mass spectrometry (Figure S1a) and HPLC (purity of >98%, see Figure S1b). ATR IR analysis of the peptide film resolves a resonance at 1654 cm−1 (Figure S1c), characteristic of amide I band of random coil structure42 in good agreement with circular dichroism studies (data not shown). The film faradaic response has an associated half-wave potential (Ein) of (0.360 ± 0.002) V, a peak separation of ∼10 mV and an expected linear dependence of peak current on voltage scan rate (see Figure S5). Molecular surface density was determined by three independent methods (Table S1) to have a mean of 2.13 ± 0.70 × 10−10 mol cm−2, in good agreement with prior reports for cysteine-anchored peptide SAMs.43−48 The capacitive redox activity of the interface as evaluated by impedance-derived capacitance spectroscopy (Nyquist resolved capacitance) is shown in Figure 1b. Unless explicitly indicated, measured values reported correspond to a mean as resolved across three independent electrodes with associated errors reported as the standard deviation of the mean. A resolved Cr value of 267.0 ± 7.1 μF cm−2 at Ein is markedly greater than either the capacitance of bare gold (11.0 ± 0.8 μF cm−2) or the SAM at redox out potentials (potentials where no faradaic redox activity is observed, Eout; 9.0 ± 0.7 μF cm−2). In utilizing this redox signal as a transduction of any subsequent binding, it is imperative that its stability is both assessed and deemed adequate. The films utilized herein display a signal output that changes
