Optimized Diagnostic Assays Based on Redox Tagged Bioreceptive

Article pubs.acs.org/ac

Optimized Diagnostic Assays Based on Redox Tagged Bioreceptive Interfaces Flavio C. Bedatty Fernandes,† Amol V. Patil,‡ Paulo R. Bueno,*,† and Jason J. Davis*,‡ †

Institute of Chemistry, Physical Chemistry Department, Nanobionics group, Univ. Estadual Paulista (São Paulo State University, UNESP), CP 355, 14800-900, Araraquara, São Paulo, Brazil ‡ Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3QZ, United Kingdom S Supporting Information *

ABSTRACT: Among the numerous label free electronic biomarker assay methodologies now available, impedance based electrochemical capacitance spectroscopy (ECS), based upon mapping the perturbations in interfacial charging of redox elements incorporated into a biologically receptive interface, has recently been shown to be a convenient and highly sensitive mode of transduction and one which, additionally, requires no predoping of analytical solution. We present, herein, a data acquisition and analysis methodology based on frequency resolved immittance function analysis. Ultimately, this enables both a maximization of assay sensitivity and a reduction in assay acquisition time by an order of magnitude.

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phene.22,23−25 FET based sensors, given the innately environmentally sensitive electrical characteristics of a 1D wire and advances in fabrication and layout (though this remains demanding), have been used to develop sensitive specific sensors with multiplexing ability.26−28 EIS assays (faradaic or nonfaradaic), based on the electrical impedance of an interface probed under a constant DC bias potential, are uniquely nondestructive, topologically flexible, and sensitive to changes at a receptive interface without requiring any amplification or labeling. It has been extensively utilized in the quantification of protein markers, including those in real patient samples.9,29−32 In its ubiquitous faradaic form, the analytical solution is predoped with a large excess of “redox probe” and an associated charge transfer resistance (Rct) (typically obtained by fitting acquired impedance to a Randles equivalent circuit model) is used as a sensing parameter.33−36 In the absence of a solution phase redox probe, nonfaradaic analyses utilize the modulus of impedance (|Z|), double layer capacitance (Cdl), or phase (ϕ) as sampling functions.37−41

he detection and quantification of protein biomarkers in complex biological samples lies central to proteomics, early clinical diagnosis, effective therapeutic application, and tracking of pharmacological response to therapeutic intervention.1−3 Current antibody based optical microarrays are commonly based on sandwich assays in which antigen binding to the immobilized antibody is detected through the use of a secondary labeled antibody. Though recent technological improvements have made such approaches more sensitive, and even multiplexed, they remain rather laborious, associated with multiple variables, and in need of a specifically labeled secondary antibody (with an assumed constant reporting output) for every antigen of interest.4,5 Direct target labeling protocols are potentially perturbative and time-consuming and can lead to high background signals. Commonly applied label free detection assays, such as those based on plasmon resonance or mass, typically offer adequately sensitive detection limits only and require the use of sophisticated and expensive pieces of equipment.6−8 In the quest to develop next generation biomarker assays, electrochemical methods remain a popular focus due to their inherently favorable attributes of high innate sensitivity, facile miniaturization, and microfluidic integration and the ability to simultaneously quantify multiple biomarker targets on chip with potentially very minimal user intervention.9−12 A broad range of electrochemical methods, including those which are voltammetric (including linear sweep, differential pulse, and square wave), amperometric, or electrochemical impedance spectroscopy (EIS) (faradaic or nonfaradaic) based have been applied to biomarker determination.12−20 A typical electrochemical sensor may map, for example, changes in measured current/potential arising from the presence of an analyte of interest, very often utilizing a labeled secondary antibody with, for example, signal amplifying nanoparticles 21 or gra© XXXX American Chemical Society

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REDOX TAGGED INTERFACES Traditional EIS measurements monitor biorecognition events at the surface of a suitably modified electrode with solution based redox reporter, utilizing as noted, Rct as the principal analytical parameter. In incorporating the redox reporter within the bioreceptive layer (for example, by using mixed selfassembled monolayer containing ferrocenethiol), we have recently reported an impedance derived methodology in which the target binding event induces perturbations in redox capacitance signal (Cr) enabling the construction of sensitive Received: August 4, 2015 Accepted: November 19, 2015

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DOI: 10.1021/acs.analchem.5b02976 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry analytical curves.42 Cr, which inherently possesses dielectric and quantum characteristics, is obtained graphically by generating a Nyquist plot and monitoring the value of the real component of complex capacitance (C′) when the imaginary component of complex capacitance (C″) has minimal contribution.12,43−45 This approach, termed electrochemical capacitance spectroscopy (ECS), represents a powerful and distortion-free means of analyzing the redox and electrostatic characteristics of any conductive surface modified with an electroactive film and can be applied to biosensing by integrating target recruiting moieties within the same film.44,46−49 ECS, to its advantage (particularly in comparison with traditional EIS methodology), obviates the need to predope the analytical solution with a massive excess of amplifying redox probe/label, and it does not require “equivalent circuit” analysis, making it particularly attractive for facile multiplexed quantification. Prior to any capacitive analysis within such films, it is useful to apply cyclic voltammetry to the redox confined molecular film in order to analyze the faradaic characteristic and identify the applied bias windows of maximum redox activity (i.e., the half wave potential E1/2) and redox inactivity (Eout) (Figure 1a),43,48,50 thus correlating with maximal and minimal capacitive activity. EIS data is then acquired over a large frequency range (typically 1 MHz to 0.01 Hz) within both potential windows and then converted to capacitive data (C* = 1/jωZ*). By subtracting the values of C′ and C″ measured at Eout from E1/2, it is possible to isolate the redox contribution to interfacial charging; Cr is specifically defined at this point from the diameter of the semicircle in the Nyquist capacitive plot as shown in Figure 1b.51 It has been shown to be particularly sensitive to interfacial change, reporting directly on redox site occupancy.44,45,50 If one considers a single redox site energy level, this is given by Cr = e 2 Γ

df e2 Γ = f (1 − f ) dμe kBT

(1) Figure 1. (a) A typical cyclic voltammogram obtained from a redox tagged mixed self-assembled monolayer composed of 11-FcC/ 16MHDA (50:50) used to determine E1/2 and Eout potential values. (b) Nyquist capacitive plot (C″ versus C′) obtained for the receptive film (biosensor) upon immobilization of anti-CRP on the mixed monolayer. As mentioned, ECS data was acquired at two potential values: E1/2 and Eout, prior to conversion of raw impedance to capacitance data. Thus, Cr, the diameter of the semicircular plot (indicated by the black arrow), is obtained by the subtracting Eout capacitance values from those acquired at E1/2.

where f = (1 + exp[Er − μe/kBT])−1, e is the elementary charge, kB is the Boltzmann constant, Γ is the redox molecular surface coverage, T is the absolute temperature, Er is the redox potential of the redox tagged element, and μe is the electrochemical potential of electrons in the electrode. Note that in eq 1, for a given system, all terms are constant except the energy/occupancy level (statistically and energetically controlled by f). This is, of course, maximized at the half-wave potential (i.e., f = 1/2).43,50 It can also be noted that the inverse function, i.e., 1/Cr has typically been used as a transducer signal and is proportional to the energy of the redox tagged interface.45−48,52 In summary, there are two important points to be noted. First, the redox capacitance signal is not the common electrostatic capacitance whose magnitude depends exclusively on electrode dimensions, but a capacitance whose magnitude depends on the redox energy levels and their occupancy within the molecular film.44,45 This in turn reflects the chemical energy of the interface and thus can potentially report on any neighboring recognition event. Second, nonfaradaic and redox contributions to the redox capacitance magnitude can be deconvoluted by acquiring data at Eout and E1/2.

resulting in a single analyzing parameter (in the case of faradaic EIS, this is Rct, a term generated by fitting data to an equivalent circuit; for ECS, a graphical analysis of data at two different applied potentials generates Cr). Both of these approaches, to varying degrees, can be rather laborious, particularly when one is seeking to develop a statistical analysis of a large volume of acquired data (over, for example, a patient cohort). In seeking to overcome this, we have recently reported the use of a portfolio of mathematically derived immittance functions (ImFs) (used as analytical parameters), based on the use of electrical transfer characteristic of the impedimetric signal as a function of frequency.53 This approach enables the use of a significantly smaller data acquisition time, provides much greater analytical flexibility, and maximizes assay sensitivity.53 The method, in a general sense, tracks perturbations across a library of frequency-dependent functions as a function of target

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IMMITTANCE FUNCTIONS AS ANALYTICAL TOOL The default approach, for both faradaic EIS and ECS, is a timeconsuming data acquisition over a wide frequency range B


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Analytical Chemistry

NaH2PO4·12H2O, 0.2 g L−1 KCl, and 0.2 g L−1 NaNO3. Polyethylene glycol (PEG) containing thiol HS−(CH2)11− (EG)3−OCH2−COOH (PEG thiol) was purchased from Prochimia Surfaces, Poland. 16-Mercaptohexadecanoic acid (16-MHDA) and 11-ferrocenyl-undecanethiol (11-FcC) were purchased from Sigma. All other chemicals were of analytical grade. Deionized water (18.2 MΩ/cm, Synergy Ultrapure water system EMD Millipore) was used throughout. Apparatus. Electrochemical experiments (cyclic voltammetry and EIS) were conducted with an Autolab Potentiostat equipped with a frequency response analysis (FRA) module using a three-electrode system: conventional gold disk working electrodes (1.6 mm diameter (BASi) or 2.0 mm diameter (Metrohm)) with a platinum wire counter electrode and a silver/silver chloride (Ag|AgCl, filled with 1.0 M KCl, +0.236 versus SHE) reference electrode (CH Instruments). All potentials reported are relative to the Ag|AgCl reference. EIS responses were recorded over a wide range of frequencies from 0.01 Hz to 1.0 MHz (at logarithmically spaced values). The reported error bars are the standard deviations of three successive measurements at any given concentration at the same electrode. In addition, each assay was repeated 3 times (across 3 independent electrodes) in order to verify the observed pattern in the sensitivity of ImFs parameters. Presented data is from a typical electrode response (averaged over 3 repeated runs) Fabrication of Receptor Interface. Gold disk electrodes were mechanically polished to flat, mirror-like surfaces (with diamond sprays of decreasing size: 1, 0.25, and 0.1 μm on polishing pads). This was followed by rinsing and ultrasonic washing in deionized water and absolute ethanol. The electrodes were then immersed in hot piranha solution (concentrated H2SO4/30% H2O2, v/v 3:1; Caution: piranha reacts extremely aggressively with organic materials. Please treat with extreme care!) for approximately 15 min, followed by rinsing and ultrasonic washing with deionized water for 1−2 min. Electrode surfaces were then electrochemically polished as follows: initially, a number of cyclic voltammetry scans (from −1.7 to −0.7 V) were run in 0.5 M KOH aqueous solution, until reproducible, stable curves were obtained. Subsequently, a series of wider potential range scans (−0.1 to 1.4 V) were conducted in 0.5 M H2SO4 at a scan rate of 0.1 V/s, until the height and shape of anodic and cathodic peaks were constant. The redox tagged bioreceptive interfaces were generated by immersing freshly cleaned gold electrodes in a mixed ethanolic (HPLC grade) solution of 1.0 mM of 16-MHDA and 1.0 mM 11-FcC (50:50%) for 16 h. Prior to CRP antibody or α-sync immobilization, the surface modified electrodes were rinsed with absolute ethanol and dried in a flow of nitrogen gas. The terminal carboxyl groups were then activated with 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide (EDC) (0.4 M) and Nhydroxysuccinimide (NHS) (0.1 M) in deionized water for 40 min and then reacted with 1 μM of each respective receptor molecule in PBS solution for 1 h, at room temperature.12,65 The interfaces were then immersed in 1 M ethanolamine (pH ∼ 8.5) to deactivate any unreacted activated carboxylic groups and washed with PBS prior to measurements. Measurements. Cyclic voltammetry and impedance derived capacitance analysis were carried out at each step of interface modification. Each final functionalized surface was exposed to its specific target at concentrations, in PBS, ranging from 102 to 104 pM (α-sync antibody) and from 10 to 105 pM (for CRP), while all impedimetric and cyclic voltammetry

concentration. The basis of this treatment is, in the first instance, an acknowledgment of EIS as a specific form of a transfer function in the case of voltage and current input/ output signals. From the applied modulating potential [V(t) = V̅ + Ṽ ejωt] and the resulting sinusoidal current response [I(t) = I ̅ + Iẽ j(ωt−ϕ)], a number of “interdependent” functions, termed as immittance functions ImF(ω) can be derived without any reference to an assumed equivalent circuit or physical chemistry picture. Each of the ImF functions can be used as an analytical signal with further possibility of optimization based upon an analysis of function sensitivity across different frequencies. The application of this methodology was recently reported by us within otherwise experimentally standard faradaic and nonfaradaic protein detection assays.53 We herein apply this within redox capacitance assays of two clinically important biomarkers, select optimal transduction parameters, and dramatically decrease analytical time. The sensitivity of α-synuclein (α-sync) antibody and Creactive protein (CRP) assays based on redox active/tagged interfaces was compared by using standard ECS and immittance function ImF(ω) methodologies. α-sync is a small (14.4 kDa) protein comprising 140 amino acids and predominately expressed in neural tissue.54 In healthy individuals, it plays an essential role in synaptic transmission and synaptic plasticity by augmenting transmitter release from the presynaptic terminal.55 However, the dysfunctional regulation of α-sync is viewed as a key factor in the pathogenesis of Parkinson’s disease (PD) as its misfolding, aggregation, and fibrillation is primarily responsible for the generation of Lewy bodies.56,57 The resulting host autoimmune response to this process has generated considerable interest, and several studies have reported correlation between quantified α-sync autoantibody levels and PD disease status.58−61 C-reactive protein (CRP), the second biomarker studied herein, is an important reporter of potential cardiac events and inflammation12 where its quantification can be an indispensable early warning assay. Inflammation, caused by infection or injury, can lead to a dramatic (1000-fold) increase in the CRP level,62 and recent work has suggested that levels exceeding 30 nM (3.0 mg/L) in serum are indicative of the risk of diabetes, hypertension, and cardiovascular disease.63,64 Herein, we have utilized mixed redox tagged self-assembled monolayers modified with α-sync or CRP antibodies as receptive and redox charging surfaces capable of specific target recruitment. Having acquired the raw impedance data (typically, over the frequency range that spans from 1 MHz to 0.01 Hz; at Eout and E1/2), we analyze the redox capacitance (Cr) and used its inverse (1/Cr) as a standard analytical signal. The acquired raw impedance data was then additionally treated to compute the immittance functions ImF(ω), namely, Z*, C*, Y*, and M* which were quantitatively analyzed in terms of real (ImF′) and imaginary components (ImF″), absolute modulus (| ImF|), ratio (ImF′/ImF″ or inverted ratio), and the inverse of each function (1/ImF).

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EXPERIMENTAL SECTION Chemical Reagents. Ethanolamine (98%), 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimde (NHS), human CRP polyclonal antibody, human CRP, hPAP antibody, and hPAP were purchased from SigmaAldrich while anti-α-sync and α-sync were purchased from Santa Cruz Biotechnology. Phosphate buffered saline (PBS, pH 7.4) contained 8 g L−1 NaCl, 0.2 g L−1 KH2PO4, 1.15 g L−1 C


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inherent mathematical correlation, each is a reporter of interfacial change and can be analyzed independently in arriving at the most sensitive and frequency optimized linear trend with target protein concentration. We evaluated the relative response (RR) of each ImF to the respective analyte/target systematically across the full frequency range where the relative response (RR) was defined as

measurements were recorded in a supporting electrolyte of 20 mM TBAClO4 (tetrabutylammonium perchlorate) dissolved in acetonitrile and H2O (20:80 v/v). A retention of binding efficacy and specificity of the biointerface in this medium was demonstrated by the self-consistency of response to specific antigen in repetitions, the consistent lack of nonspecific response to BSA, and prior reports.47,52,66 CV analyses were performed at a scan rate of 100 mV s−1 between 0.0 and 0.7 V relative to Ag|AgCl. On the basis of the cyclic voltammetry data, half-wave potential E1/2 and Eout were determined to be around 0.45 and 0.1 V vs Ag|AgCl reference electrode, respectively. EIS measurements were conducted in a frequency range of 0.01 Hz to 1 MHz with RMS amplitude of 3 mV (or 10 mV peak to peak) at E1/2 and Eout for each of the measured target concentrations. Limits of detection (LoD) were calculated, on the basis of the standard deviation (SD) of the blank response and slope of the analytical curve, as 3.3 times SD. The specificity of each functionalized surface was tested by exposure to equivalent concentrations of BSA. In all cases, this was accompanied by negligible nonspecific binding (see Supporting Information S-1). An extension of the outlined methodology to specific detection of a target antigen in surrogate animal serum is absolutely demonstrable (see Supporting Information S-2) but not the subject of this methodological paper.

RR nω(%) = [(R nω − R 0ω)/R 0ω] × 100

where is the initial value of ImF function in absence of analyte and Rωn is the value of the ImF function after exposure to a specific target concentration (n) at the same angular frequency (ω). Thus, for any given immittance function, it is possible to define a particular frequency or a frequency range within which the relative response (RR) is optimal. A home written MATLAB R2014b algorithm was used to calculate and convert the original electrical transfer function data into 28 phasorial related immittance functions and to determine the optimal frequency at which the response of ImF is monotonic and of good linearity (with r2 > 0.96). Only those functions that showed favorable response (in terms of monotonic behavior and linearity/range of response) were further analyzed while the rest were discarded. Thus, by analyzing the receptivity of each functionalized surface (for αsync antibody and CRP) for each of the 28 ImF functions using raw impedance data sets (acquired at E1/2 and Eout), it is possible to compare the optimal sensitivity of any ImF function with the standard redox capacitance signal (Cr). It is important to note that all of the ImF parameters measured at Eout either showed a poor linearity (r2 > 0.9), i.e., poor response, or extremely low responsiveness to the target (less than 10% per decade of the target concentration) and were accordingly discarded from further consideration. The same functions analyzed at E1/2 were associated with sensitivities exceeding 100% per decade of the target concentration combined with good linearity (r2 > 0.96) (see Supporting Information S-3). This initial observation confirms the specific high sensitivity of the redox capacitive component (with its dominant quantum contribution)44,45 to environmental change. Indeed, the electrochemical capacitive signal associated with the faradaic activity is highly sensitive when compared with nonfaradaic capacitive signal component due to its intrinsically highly localized “electronic” characteristics. Note that nonfaradaic charging, intrinsically ionic in nature, is relatively very unresponsive.48,49,53 Taking the frequency response of 1/C″, as a typical representative immittance function, for α-sync antibody detection (Figure 2), it is evident that RR varies across the frequency range with a large apparent change in magnitude (i.e., high sensitivity) within a limited frequency range of 10 to 120 Hz and low response elsewhere (see the Supporting Information document for additional data based on CRP). By computing the averaged RR response (for three measurements on the same functionalized surface at each concentration of target) across the concentration range (102−104 pM for α-sync antibody and 10−105 pM for CRP) and fitting a linear response (r2 > 0.96) if monotonic, it is possible to determine the optimal frequency for each of the 28 sensitive parameters at which the sensitivity (S) (defined as the slope of the linear fit) is maximal; see Supporting Information Table 2. In comparing the performance and optimal analyzing frequency of each of ImF parameter and subparameters, for both targets, responses were found to be maximal for 1/C″.

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RESULTS AND DISCUSSION While the acquisition of raw impedimetric data, at two different potentials, in generating Cr, is straightforward, it does require significant data acquisition time (∼800 min for a typical analytical curve consisting of individual measurements at eight concentration analyte values). In seeking to bypass the multiple large frequency range data acquisition and analysis, we have utilized the spectroscopic data processing and optimization methodology [ImF(ω)] recently outlined and summarized below.53

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IMMITTANCE FUNCTION ANALYSIS The analyzed immittance complex functions (ImF*), as computed from the raw impedance data (acquired at applied bias of E1/2 and Eout) were impedance (Z*), capacitance (C*), modulus (M*), and admittance (Y*) with their phasorial relationships. V ̃ e jω t I ̃ e(jωt − ϕ)

(2a)

C* = 1/(jωZ*)

(2b)

M * = jωZ*

(2c)

Y * = jωC*

(2d)

Z* =

(3)

Rω0

where j = −1 and the angular frequency is defined as ω = 2πν with the linear frequency ν given in s−1 (Hz). Note that the asterisk denote complex function. As complex functions, each of these can be individually divided into constituent real (ImF′), imaginary (ImF″), inverse (1/ImF′ and 1/ImF″), absolute modulus (|ImF|), ratio (ImF′/ ImF″), and inverted ratio (ImF″/ImF′) forms, each of which can be used analytically.53 A total of 28 parameters for any given sampling frequency (Supporting Information Table 1) were, then, available from the raw impedimetric data. Although some functions have an equivalent response due to their D


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Figure 2. A typical example showing the change in the relative response (RR) of the 1/C″ parameter obtained by treating the raw E1/2 impedance data of a α-sync antibody ECS assay on a α-sync modified 16-MHDA/11-FcC gold electrode; as concentration was varied from 102 to 104 pM. The change in RR is maximal around ∼40 Hz. Figure 3. Comparative analytical responses on 16-MHDA/11FcC mixed films appropriately modified for (a) α-sync antibody and (b) CRP detection. It is evident that, in both assays, 1/C″ is more sensitive than 1/Cr as indicated by the slope of the assays (S). The optimal analytical frequencies for the 1/C″ parameter are 40.47 Hz (α-sync) and 21.06 Hz (CRP). Standard deviation is calculated across 3 measurements for each given concentration with all r2 > 0.99.

In analyzing trends in more detail, we see that, across a linear range of 102−104 pM for α-sync antibody, the most responsive linear parameters found were 1/C″, C′/C″, C′, Z′, and Z″ (a trend consistent across three equivalently prepared electrodes) with sensitivities of 159.1 ± 2.7, 106.2 ± 2.4, 105.7 ± 2.0, 105.3 ± 1.6, and 104.8 ± 4.3% per decade of concentration, respectively. Corresponding LoDs are 121.1 ± 2.7, 118.9 ± 3.1, 146.8 ± 6.1, 105.7 ± 2.2, and 143.6 ± 6.5 pM, respectively (Supporting Information Table 2). These analytical criteria, which are comparable with the 1/Cr data (sensitivity of 84.0 and LoD of 143.5 ± 9.7 pM) (Figure 3a), require no Nyquist/ Bode graphical analysis. For CRP assays, a linear range of 10 to 105 pM (0.001 to 10.0 mg L−1, covering the useful clinical range as defined by American Heart Foundation and United States Center for Disease Control67) was obtained for all ImF parameters. In addition, out of all the available immittance functions, 1/C″, Z′, C′, C′/C″, and Z″ were found to be optimal (again, a trend consistently observed across equivalently prepared electrodes) showing sensitivities and LoDs of 70.8 ± 1.6, 41.7 ± 2.1, 38.7 ± 1.8, 37.6 ± 0.9, and 31.2 ± 3.1 and 7.4 ± 3.1, 0.48 ± 0.2, 9.5 ± 2.8, 3.4 ± 1.3, and 4.9 ± 2.2 pM, respectively. These values, once again, are comparable to 1/Cr as a reporting function (sensitivity of 30.4 ± 0.7, LoD of 5.4 ± 1.6 pM) (Figure 3b), and all represent an assaying prowess which is highly competitive with existing labeled methods as applied to this clinically important marker.68 It should be further noted that the response and resulting sensitivity of the capacitative interface depends upon a number of parameters; the first of these is the magnitude of the transducing redox capacitance signal. This depends, in turn, upon the density of states (DOS) of the surface confined redox centers.43−45,50 Target capture efficacy will be, as always, dependent on appropriate antibody exposure to solution and its surface density.12 Assay sensitivity is also dependent, of course, on the degree to which target capture is transduced by the measured signal. In this case, this represents the degree to which the local electrostatic/dielectric environment of the

ferrocene centers responds to capture at a neighboring antibody surface. Within this present “proof of concept”, we have utilized a fixed and equimolar film composition. Unquestionably, one can seek substantial sensitivity and indeed selectivity improvements by tuning film preparation and composition and/or the use of high surface area supports.69 The real strength of ImF analysis of ECS data, and the subject of this report, lies not in the development of assays that are equal (if not superior) in sensitivity to more standard redox capacitive assays but more so in the signif icant reduction in time required for data acquisition and subsequent analysis. The acquisition of a seven point analytical calibration curve, with triplicate repeats at each defined concentration, requires 700− 800 min by redox capacitance (ignoring the incubation time required for each concentration). An initial change to ImF analysis requires data acquirement at just one surface potential, so this timeline is immediately halved. In screening acquired impedimetric data, it, additionally, becomes apparent that there exists a frequency range, around the optimal frequency, for which assay sensitivity is largely equivalent to maximal sensitivity. Figure 4a, for example, shows a broadly constant sensitivity to α-sync antibody, as monitored by Z″ (chosen as a typical representative ImF function), across a frequency range of 13 to 5000 Hz (with an average sensitivity value of 100.1 ± 4.9 as measured over 15 data points; this can be compared against the maximum sensitivity value of 104.7 at 2423.0 Hz). The existence of a consistent broad optimal frequency range enables users to scan over a limited frequency range without requiring prior knowledge of the exact value of the optimal f requency and yet attain assays with comparable E


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Figure 5. Comparative ImF parameter sensitivities for α-sync antibody (a) and CRP (b), showing the existence of an equivalent frequency range, as indicated by the f requency error bars, within which sensitivities are comparable to optimal values (i.e., highest sensitivity at a single optimal frequency). It is evident that parameters like 1/C″ and Z′ are the most sensitive and confined with a frequency range of 0.1 to 100.0 Hz.

Figure 4. (a) Optimal analytical curve of redox capacitance analysis of α-sync antibody on mixed film composed by 16-MHDA/11FcC modified gold electrode showing that Z″ has similar optimized relative sensitivity between 13 and 5000 Hz (dashed black line) (sensitivity of 100.1 ± 4.9 against the maximum sensitivity of 104.7, found at 2423.0 Hz, r2 = 0.980 ± 0.009). It should be noted that data acquisition time for a limited frequency range (13 to 5000 Hz) is about 15 s while about 17 min is required for a full frequency range scan! (b) A summary of relative ImF parameter sensitivity in a redox capacitance α-sync detection assay (102 to 104 pM). Data here was obtained by averaging across an optimal frequency range of 13−5000 Hz, and the analytical curve was constructed grouping the values for each concentration at different frequency within the given range of 13 to 5000 Hz.

means that the total data acquisition time (acquisition steps required to generate a 7 concentration point analytical curve based upon triplicate measurements at each concentration) would now be reduced to approximately 20 min (Figure 6). The enclosed work has, then, established a means of optimizing assay time and sensitivity for a given film

sensitivity, LoD, and linear range with a further notable reduction in data acquisition time. Figure 4b, for instance, represents the analytical curve constructed by selecting the average response and SD for each analyte concentration measured within the frequency range (of 13−5000 Hz). Clearly, the signal variation for each concentration of the target is minimal (error bars less than 2.5% in terms of relative standard deviation, RSD; data representative of 1 electrode with three repetitions), while limiting the total calibration acquisition time to ∼23 min. A detailed analysis of optimal frequency range for some of the ImF’s parameters in redox capacitive assays of α-sync antibody and CRP is presented in Figure 5. It is worth noting that, for both targets, ImF optimal response/sensitivity is confined to the 0.1−100.0 Hz frequency range for the best parameters found herein (1/C″ and Z′). However, for C′/C″, Z″, and C′ (which also showed excellent analytical response), the optimal sensitivities were found at higher frequencies (∼103 up to 104 Hz) still enabling substantially shorter data acquisition times. From a practical perspective, an ability to confine the typical sampling frequency range (initially mapped from 0.01 Hz to 1 MHz) to a much smaller frequency window

Figure 6. Any assay is typically represented by an analytical curve (as shown in Figure 3). However, immittance function data analysis shows that, while the sensitivity(s) (as calculated from the analytical curve) is better if not equal to 1/Cr, the time required to acquire the data for immittance function analysis, over a limited frequency window, is smaller by an order of magnitude as indicated in the bar plot. F


Analytical Chemistry composition. As noted above, there are very good grounds to believe that sensitivity can be improved further by tuning the film composition.12,43,44 It is worth noting that, even without this, the two markers analyzed here are resolved with detection limits and linear ranges that are directly relevant to real clinical analyses; previous electroanalytical assays of α-synuclein autoantibodies have reported, for example, concentrations in the 0.1 to 3 nM (within the 0.1−10 nM linear range herein) range as being diagnostically differentiating.29,70 In the case of CRP, serum levels of less than 8 nM are considered to be normal, while levels greater than 25 nM are indicative of cardiovascular diseases.64 The redox capacitative assays herein both comfortably match this range and do so with sensitivity that compares favorably with previous impedimetric assays of this marker.12,18,71,72

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CONCLUSIONS

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ASSOCIATED CONTENT

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ACKNOWLEDGMENTS

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REFERENCES

We acknowledge São Paulo state research funding agency (FAPESP) for the financial support granted to F.C.B.F., Process number 2015/13359-2.

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The development of reliable, sensitive, and selective biomarker detection assays undoubtedly brings with it potentially game changing improvements in early disease diagnosis, more effective intervention, and a tracking of patient response to treatment. While a range of methods now offer adequate sensitivity, those which are electronic are most easily integrated into platforms which offer an innate multiplexing, low cost, and high throughput application with minimal operator intervention. Label free electrochemical capacitance spectroscopy (ECS) can support high levels of detection sensitivity without amplification or solution doping. We believe the outlined methodological process is of value for both optimizing the efficacy of such assays and dramatically reducing analytical time. Unlike standard capacitive or impedimetric approaches, which require a measurement of impedimetric change across a large frequency window and at multiple potential values, the ImFs approach outlined can be applied to the same raw data acquired at just the half wave potential and enables a much more confined data volume to be utilized. This in turn reduces the time taken to acquire a seven point triplicate repeat analytical curve from ∼800 min to ∼20 min and supports a subsequent unknown quantification to be carried out (in triplicate) in ∼3 min (rather than 90 min as required by traditional redox capacitance techniques). The practical advantages of this, in terms of throughout and multiplexing, are significant.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02976.

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Specificity data in serum, Bode plots, and tabulated ImF function paramaters for both CRP and synuclein (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Phone: +55 16 3301 9642. Fax: +55 16 3322 2308. E-mail: [email protected]. *Phone: +44 1865 275914. E-mail: [email protected]. Notes

The authors declare no competing financial interest. G


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