Chronoamperometric Magneto Immunosensor for Myeloperoxidase

Article pubs.acs.org/ac

Chronoamperometric Magneto Immunosensor for Myeloperoxidase Detection in Human Plasma Based on a Magnetic Switch Produced by 3D Laser Sintering Jaume Barallat,† Rosa Olivé-Monllau,§ Javier Gonzalo-Ruiz,§ Raúl Ramírez-Satorras,§ Francesc Xavier Muñoz-Pascual,§,∥ Amparo Galán Ortega,‡ and Eva Baldrich*,§ †

Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Spain Hospital Germans Trias i Pujol, Badalona, Spain § Institut de microelectrònica de Barcelona (IMB-CNM, CSIC), Campus Universitat Autònoma de Barcelona, Bellaterra 08193, Spain ∥ MATGAS A.I.E. Campus UAB, Bellaterra 08193, Spain ‡

ABSTRACT: In this work, an amperometric immunosensor for detection of myeloperoxidase (MPO) in human plasma is reported. Detection is based on the immobilization of anti-MPO antibodies onto magnetic beads (MBs). Following MPO immunocapture and washing steps, MBs are transferred to a customized modular detector device produced by 3D laser sintering. This tool integrates electrodes, electrical connectors, and a novel magnetic switch, whose functioning is founded on the vertical displacement of a permanent magnet. In this way, magnetic switching makes possible the confinement of MBs over the working electrode for electrochemical detection, followed by the release of MBs for electrode washing and reutilization. Notably, electrochemical detection is based on the endogenous MPO activity, which reduces reagent consumption and assay time compared to sandwich assays using enzyme-labeled antibodies. After optimization, the assay could be completed in 45 min and displayed a linear response between 0.9 and 60 ng mL−1 for MPO and a limit of detection of 0.4 ng mL−1. The real applicability of this approach is demonstrated by the ability to carry out the successful analysis of MPO in human plasma samples. Furthermore, the results allowed the classification of patients into three groups at risk of suffering cardiac events (i.e., low, medium, or high) and correlated well with data provided by a commercially available standardized method.

A

superfamily. In addition to its peroxidase activity, MPO catalyzes the reaction between chloride and hydrogen peroxide to produce hypochlorous acid (HOCl), a potent antimicrobial agent that has been also involved in the pathology of atherosclerosis.4 Since systemic MPO levels are associated with coronary plaque erosion5 and increased amounts of MPOexpressing macrophages are present in eroded or ruptured plaques causing acute coronary syndrome,6 MPO is currently accepted as a marker for diagnosis and stratification of acute coronary syndrome.7−10 In contrast to the characteristics of other well-established biomarkers such as troponin and the MB fraction of creatine kinase, MPO monitoring allows identification of patients at risk of cardiac events even in the absence of myocardial necrosis, providing a new approach for early diagnosis in daily clinical practice.7−9 Most of the currently accepted reference methods for determination of MPO are spectrophotometry-based sandwich immunoassays, frequently conducted in central laboratories using robotic platforms.11,12 The alternative implementation of electrochemical immunosensors could facilitate rapid point-ofcare diagnosis and/or improve the practicability of pre-existing

ccording to the World Health Organization, nearly 30% of the 57 million deaths occurring worldwide in 2008 were caused by cardiovascular diseases (CVD).1 This places CVD as the leading cause of death in the world, higher than that of infectious diseases (>16%), cancer (13%), and chronic respiratory disorders (8%).2 CVD occurrence is favored by behavioral risk factors such as tobacco, physical inactivity, unhealthy diet, and alcohol abuse, which are estimated to be responsible for about 80% of coronary heart and cerebrovascular diseases. Accordingly, while the number of deaths attributed annually to infectious diseases are projected to decay over the next 20 years due to the improvements in sanitation and access to drugs and medical care, it is anticipated that increased longevity and the global changes in dietary consumption and lifestyle will result in an increase in the number of deaths due to CVD from 17 million in 2008 to about 25 million in 2030.1−3 This could be partially counterbalanced by the fact that pharmacological and surgical treatment of these conditions have dramatically improved over the last decades. Consequently, there is a growing demand for novel biomarkers to ameliorate prognosis, diagnosis, and follow-up of this type of patient. Myeloperoxidase (MPO) is a 150 kDa heme-containing enzyme that is produced and secreted by neutrophiles and monocytes, which belongs to the peroxidase−cyclooxygenase © 2013 American Chemical Society

Received: May 23, 2013 Accepted: September 3, 2013 Published: September 3, 2013 9049

dx.doi.org/10.1021/ac401549d | Anal. Chem. 2013, 85, 9049−9056

Analytical Chemistry

Article

which material and configuration were the most suitable for MPO determination. All of them integrate a working, counter, and reference electrode (Table 1).

methods, providing better health care and reducing turnaround time, which is highly stressful for the patient.13−15 However, and to the best of our knowledge, only a few attempts to detect MPO electrochemically have been reported to date.16−20 Biosensors consist of a physical transducer modified with a biorecognition element so that target affinity−capture directly translates into signal transduction. Their specificity relies on the physical modification and blocking of the transducer in order to guarantee response specificity and minimal levels of nonspecific adsorption of unwanted species. Nevertheless, in the case of electrodes, this might negatively affect electron transfer rate and commonly results in decreased transducing efficiency. For this reason, a number of strategies have been reported in which capture and transduction surfaces are physically separated.21−23 The most successful and extensively exploited among them has been the utilization of immunofunctionalized magnetic beads (MBs).24−26 Since incubation of MBs with the sample is usually carried out under rotation, the combination of a large capture surface area with improved sample mixing reportedly results in enhanced target−antibody kinetics, shorter assay times, and a limit of detection (LOD) improvement compared to classical enzyme-linked immunoassays (ELISA).27 Moreover, using MBs allows fast, simple, and specific preconcentration of diluted target molecules and physical separation from potential interfering agents present in complex sample matrices. In this work, we develop an amperometric immunosensor by immobilizing anti-MPO antibodies onto MBs. Following MPO immunocapture, MBs are transferred to a modular detector device produced by 3D laser sintering, which integrates electrodes, electrical connectors, and a switchable magnet. Since electrochemical detection is based on the endogenous MPO activity, reagent consumption and assay time are reduced compared to sandwich assays using enzyme-labeled antibodies. After optimization of different assay parameters such as incubation conditions, MB handling, sample matrix effect, and detection setting, the procedure is finally applied to the analysis of MPO in real plasma samples.

Table 1. Characteristics of the Five Electrode Devices Used in This Work working electrode

counter electrode 2

reference electrode

electrode

material

area (mm )

material

material

1 2 3 4 5

Au Pt Pt carbon SWNT

8.6 8.6 8.6 12.5 12.5

Au Pt Pt carbon carbon

Au Pt Ag/AgCl Ag Ag

Electrodes 1, 2, and 3 (1 × 5 cm) were fabricated at the Clean Room facility of IMB-CNM by standard photolithographic techniques. First, a thermal oxide insulating layer (1 μm thick) was grown onto a silicon wafer (4 in. diameter). It followed metallization and electrode patterning via two alternative strategies depending on the electrode material and device passivation for electrical interference prevention and chemical protection. In the case of electrode 1, titanium (10 nm), nickel (10 nm), and gold (150 nm) were serially deposited by sputtering. Then, a positive photoresin was spin-coated over the metal and was subsequently patterned using a contact mask and UV light. It followed metal wet etching in order to define the gold electrodes and the contact paths and device passivation with an epoxy resin (Ebecryl) using a contact mask. In the case of the devices 2 and 3, titanium (10 nm) and platinum (150 nm) were deposited with an electron gun system using a contact mask and an image reversal photoresist (AZ5214-E). The electrodes and contact paths were defined in this case by lift-off. The chips were next passivated by plasmaenhanced chemical vapor deposition (PECVD) of a double layer of silicon oxide (400 nm) and silicon nitride (400 nm). This passivation layer, which should provide electrical insulation and define electrode geometry, was patterned by reactive ion etching (RIE). Finally, for electrode 3 only, a thickfilm reference electrode was stamped by conventional screenprinting followed by heat curing (80 °C/30 min + 120 °C/5 min).28 Electrodes 4 and 5 were commercially available screenprinted electrodes (SPE; Dropsens, Oviedo, Spain). While electrode 4 (ref DRP-110) integrated a carbon working electrode, electrode 5 displayed a SWNT-COOH working electrode. Both incorporated a carbon counter electrode and a Ag pseudoreference electrode onto a ceramic substrate. Immunofunctionalization and Handling of Magnetic Beads (MBs). Unless otherwise stated, all incubations were performed in Eppendorf microcentrifuge tubes, at room temperature (RT), under continuous rotation at 10 rpm, and protected from light. Streptavidin-coated MBs (150 μL, 6−7 × 108 MB mL−1) were washed three times with 300 μL of PBS using a magnetic rack (Bilatest magnetic separator, Sigma Aldrich). The beads were then resuspended in 20 μL of biotinylated anti-MPO mAb (equivalent to 1−2 μg) and 30 μL of PBS and were incubated for 30 min. Next, two washing steps with PBS were made, followed by incubation with 50 μL of 2.5 mM biotin for 15 min in order to block any free biotin-binding sites. The modified MBs were washed three more times with PBS, and

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MATERIALS AND METHODS Chemical Reagents and Biocomponents. All solutions were prepared using ultrapure deionized water of resistivity not less than 18 MΩ·cm from a Milli-Q system (Millipore, Billerica, MA). Phosphate-buffer saline tablets (PBS; 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4) and streptavidin-modified magnetic beads (MB; Dynabeads M280 streptavidin, 2.8 μm φ, 10 mg mL−1) were supplied by Invitrogen (Life Technologies, U.K.). According to the provider’s description, MBs consist of polystyrene spheres evenly embedded with iron oxide and then covered with a polymer shell that prevents iron leakage. Myeloperoxidase (MPO; ref ab98926) and antimyeloperoxidase monoclonal antibody (mAb; ref Ab100975) were purchased from AbCam (Cambridge, U.K.). Bovine serum albumin (BSA), biotin, Tween 20, and potassium ferrocyanide (99.8%) were supplied by Sigma Aldrich (St. Louis, MO). Stabilized 3,3′,5,5′-tetramethylbencidine plus hydrogen peroxide K-blue reagent solution (TMB) was purchased from Neogen (Abyntek, Spain). This solution was used as received for the spectrophotometric determination of MPO activity but was diluted 1:10 with PBS for the electrochemical determinations. Electrode Fabrication and Characteristics. Five different electrode devices were tested in parallel in order to find out 9050



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Figure 1. Design of the customized 3D holder and detail of the magnetic switch. On the left side, the holder design integrating a silicon sensor. A, the base of the main body of the holder; B, the cover of the main body of the holder; C and D furnish the magnetic switch. On the right side, details of the magnetic switch mechanism.

centrifugation (2100g for 5 min), and plasma and serum were recovered, aliquoted, and stored at −20 °C until their utilization. For MPO determination, real samples were diluted 1:50 or 1:100 with PBS and were then assayed as described above. All samples were analyzed in parallel using an automated immunoassay method for MPO (RF425 Siemens Healthcare Diagnostics, Inc.) and processed on the automatic analyzer Dimension Clinical Chemistry System with a Heterogeneous Immunoassay Module (Siemens Healthcare Diagnostics, Inc.). This MPO method is a quantitative one-step sandwich immunoassay that measures MPO mass (not MPO activity). The sample (30 μL of plasma, which was automatically diluted and processed by the robotic platform) was incubated simultaneously with mAb-coated chromium dioxide particles and a β-galactosidase-labeled mAb. The particle/MPO/ conjugate sandwich that was formed was recovered by magnetic separation and washing, followed by addition of a βgalactosidase chromogenic substrate and colorimetric detection of the enzyme product. The whole assay took 30 min and displayed an analytical measurement range between 13 and 5000 pmol L−1 (equivalent to 1.95−750 ng mL−1) and a LOD of 13 pmol L−1 (equivalent to 1.95 ng mL−1, determined by the provider from the blanks plus twice their standard deviation). Spectrophotometric Measurements. Spectrophotometric measurements were performed in 96-well microtiter plates using an Ascent Multiskan EX ELISA reader (Thermo Scientific, Spain). This made possible the simultaneous detection of numerous samples and facilitated the preliminary

resuspension was carried out in 0.75 mL of PBS, 0.1% BSA, 0.02% sodium azide, to a final concentration of 1.2−1.4 × 108 MB mL−1. Storage was conducted at 4 °C. Analytical Procedure. MPO Magneto Immunoassay in Saline Solution. Immediately before their utilization, anti-MPO MBs were set to room temperature, were completely resuspended by vortexing for 1−2 min, and were washed three times with PBS. MPO was serially diluted either in PBS or in PBS supplemented with 1% of BSA (PBS−BSA). For each concentration, 100 μL of MPO was rotated for 30 min with 5 μL of modified MBs if they were to be used for spectrophotometrical detection or with 10 μL of modified MBs when prepared for electrochemical detection. MBs were then washed three times with PBS 0.01% Tween 20 (PBS−T) and were resuspended in their original volume of PBS (i.e., 5 or 10 μL). MPO Magneto Immunoassay in Real Samples. Blood plasma and serum samples for the calibrations were obtained from healthy volunteers. Pathological plasma samples were obtained from not individually identifiable leftover specimens collected from hospitalized patients with coronary ischemia in the Coronary Care Unit of Germans Trias i Pujol Hospital (Badalona, Spain) as described in the “Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens That Are Not Individually Identifiable”.29 Blood specimens were collected using vacutainer tubes containing lithium heparinate or clot activator and gel separation. Samples were processed as soon as possible by 9051



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The holder was designed using INVENTOR Software 2012 and was fabricated in polyamide by 3D laser sintering using Formiga P100 equipment (EOS, GmbH; Munich, Germany). It consists of four separated parts (Figure 1). Part A (3.5 × 2.8 × 0.8 cm) is the base of the main body of the holder. It has a socket that accommodates the edge of the silicon sensor that displays the connection pads. On top of it, a support with three holes houses spring-loaded connectors for the connection to the potentiostat. Part B (3.5 × 2.8 × 0.8 cm) is the cover of the main body of the holder. When parts A and B are clamped together, the spring-loaded connectors are pressed toward the sensor’s pads, which guarantees an efficient and stable electrical connection. Parts C (3.5 × 2.0 × 0.8 cm) and D (2.5 cm long × 0.4 cm diameter) furnish the magnetic switch. Part D, which holds a neodymium magnet 1.5 mm in diameter (Ingenieria Magnetica Aplicada, Bilbao, Spain), assembles inside a cavity in C. Here, and because of two series of protruding rims, D can reach two different vertical positions by rotating it 90°, while shifting it up and down in a screw-like fashion. This drives the magnet closer or farther from the sensor, which switches on and off the magnetic field at the surface of the working electrode. In this way, magnetic switching makes possible the confinement of MBs over the working electrode for electrochemical detection, followed by release of MBs for electrode washing and reutilization. Spectrophotometric Characterization and Immunoassay Optimization. The immunoassay was optimized on the surface of MBs and took advantage of the endogenous peroxidase activity displayed by MPO. Anti-MPO MBs were incubated with the samples under study, were washed, and were immediately submitted to detection. In this way, the procedure was simpler and shorter than a classical sandwich and, at the same time, benefitted from improved sample mixing and target preconcentration provided by MBs. Spectrophotometric Detection of MPO Activity. The preliminary assay optimization was carried out spectrophotometrically, which allowed simultaneous handling and detection of more samples. Anti-MPO MBs, prepared by modifying streptavidin-coated MBs with biotinylated anti-MPO antibodies, were incubated with increasing concentrations of MPO. Following washing, the MBs were concentrated using a magnetic rack, the supernatants were transferred to the wells of a microtiter plate, TMB was added, and absorbance was monitored at 620 nm for 75 min with gentle stirring every 15 min to prevent MB sedimentation. In these experiments, MPO activity resulted in an increase in A620 over time until the signal reached a plateau at around 45 min. Alternatively, the reaction was stopped at different times by adding 0.1 M HCl, and absorbance was measured at 450 nm. Under such conditions, while the negative controls remained low, the signals registered in the presence of MPO were 2 to 3 times higher than those registered at 620 nm. Optimal performance was observed by acid treatment after 30 min of reaction, when the best signal-to-noise ratio and reproducibility were obtained. Notably, the enzymatic activity of MPO was significantly lower than the activity exhibited by similar concentrations of other enzymes. For instance, the activity of horseradish peroxidase (HRP) was 78 times higher than that of MPO under similar experimental conditions (data not shown). This could be partially explained considering the differences in the accessibility of the active sites of both enzymes.30

optimization of the experimental conditions, which included assay parameters such as incubation time, sample dilution, volume of MBs per sample, sample volume, and matrix effect. After the immunoassay, the MBs were transferred to clean tubes, 100 μL of TMB was added, and the beads were rotated for 45 min in the dark. The reaction was then stopped by addition of 100 μL of 0.1 M HCl, the MBs were concentrated using a magnetic rack, and 100 μL of the supernatant was transferred to the wells of a microtiter plate. Absorbance was immediately read at 450 nm. Electrochemical Measurements. Electrochemical measurements were carried out using a computer-controlled CH Instruments 1030A multipotentiostat. All experiments were performed into a Faraday’s cage, at room temperature, and in dark conditions. Before each experiment, electrodes were cleaned successively with 95% ethanol, deionized water, and isopropyl alcohol, and they were then dried with nitrogen. Au electrodes, carbon SPE, and SWNT-COOH SPE could be used as received without any additional treatment. Pt microelectrodes were activated electrochemically by repeated steps (200 times) of 5 s each, alternating between 0 and −2 V in PBS. Electrodes were next characterized by cyclic voltammetry (CV) in 0.1 M KCl and 1 mM K4 Fe(CN)6. The optimal potential for TMB reduction at each type of electrode was determined by CV from −0.2 to 0.5 V at 50 mV s−1. For the chronoamperometric measurements of MPO in solution, 50 μL of TMB solution was placed onto the electrode, followed by signal stabilization at the potential of choice. Then, the measurement was paused, MPO was added to reach a final concentration between 0.1 and 100 and ng mL−1, the solution was mixed by pipetting, and the measurement was resumed. The whole procedure was repeated for each point of the calibration plot to obtain completely independent replicates. For the chronoamperometric measurements of immunocaptured MPO, the modified MBs were confined on the working electrode surface by switching on the holder magnet, a drop of 50 μL of PBS at pH 7.4 was added, and chronoamperometry was performed in order to establish a stable baseline. Then, the measurement was paused, the PBS solution was removed, it was replaced by 50 μL of diluted TMB solution, and the measurement was resumed. Data Analysis. The data shown come from no less than three independent replicates, and the error bars correspond to the standard deviation of those measurements. The limits of detection (LOD) and quantification (LOQ) were calculated from the average of the blanks (assays carried out in the absence of the target molecule) plus 3 or 10 times their standard deviation, respectively.

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RESULTS AND DISCUSSION Design and Fabrication of a Modular Holder. Implementation of the electrochemical magneto immunoassay reported here relied on the design and fabrication of a customized holder. This had to provide efficient connection of the electrodes to the potentiostat, as well as appropriate exposure of the electrodes to the MBs. Simultaneously, it should incorporate a switchable magnetic field. When the magnetic field was connected, MBs would be confined over the working electrode. By turning it off after sample electrochemical detection, MBs would be released for fast and simple reutilization of the electrode. 9052



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Figure 2. Immunoassay optimization by spectrophotometric detection of MPO activity. Comparison of signals obtained (a) after different sample incubation times with MB, (b) for different sample volumes, (c) using increasing amounts of MB, and (d) for different dilutions of the same serum matrix.

Optimal Immunocapture Time. Immunocapture times of 15−60 min were next compared. Different concentrations of MPO were incubated in parallel with 5 μL (1.2−1.4 × 108 MB mL−1) of either anti-MPO MB or negative control MB (with no antibodies) in order to discriminate between signals generated by specific immunocapture and by nonspecific adsorption. For the concentrations of MPO tested, the signals registered at the negative control MB did not increase significantly with incubation time, indicating that low levels of MPO nonspecific adsorption were generated onto the MB surface. When using anti-MPO MB, immunocaptures shorter than 30 min failed to detect MPO concentrations below 0.1 μg mL−1. Simultaneously, incubations longer than 30 min generated higher but more variable signals. Since MPO is a dimeric protein, this could be due to MPO simultaneous binding by more than one MB. As a result, a certain level of MB aggregation would happen during long incubations that could negatively affect detection of the enzymatic activity (Figure 2a). In accordance with these results, immunocaptures of 30 min were selected as the optimal compromise solution. Effect of Sample Volume and Incubation Conditions. In order to determine the effect of sample volume and mixing, incubation with anti-MPO MB was alternatively carried out in Eppendorf tubes or in microtiter plates. In the first case, immunocapture took place under physical rotation, which promoted complete mixing of sample and MB and prevented MB sedimentation. In the second case, microtiter plates were agitated using an orbital shaker, which mixed sample and MB but did not avoid MB settling. In both cases, sample volumes of 50−150 μL were assayed.

Incubation using microtiter plates made possible simultaneous handling of up to 96 samples using an appropriate magnetic rack and multichannel pipettes. Hence, assay execution was easier and faster. On the other hand, the assay linear range shifted toward higher MPO concentrations and poorer LOD, and lower result reproducibility was obtained. In the case of assay operation in tubes, better resolution and reproducibility were obtained for detection of low MPO concentrations, together with generation of higher signals, higher sensitivity, and lower LOD. This was attributed to the better mixing/homogenization of the sample and MB, which presumably resulted in enhanced immunocapture. On the contrary, saturation was observed at lower MPO concentrations. Since a LOD as low as possible and optimal assay performance at low MPO concentrations would better-fit the requirements for real sample monitoring, assay implementation was done in Eppendorf tubes. With regard to sample volume, optimal assay performance was obtained for incubations in 100 μL. Volumes of 50 μL showed greater intra-assay variability, probably because of a deficient mixing of the sample with the MB. Volumes of 150 μL, on the other hand, displayed wider physical dispersion in the tubes during rotation, which negatively affected MB recovery and assay reproducibility (Figure 2b). Effect of MB Load. The effect of MB load on immunocapture efficiency was studied by detecting in-parallel MPO serial dilutions using increasing amounts of anti-MPO MB (2.5, 5.0, 7.5, and 10 μL, 1.2−1.4 × 108 MB mL−1). As shown in Figure 2c, the assay improved with the MB load. The best results were achieved with a volume of 10 μL of MB, which 9053



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produced a wider assay linear range and a steeper slope of the calibration curve at low concentrations (i.e., higher assay sensitivity). Higher MB loads were not assayed in order to limit the cost of the assay per sample. Interference by Real Sample Matrix and Optimal Sample Dilution. The optimized assay took about 75 min, including rotation for 30 min of 100 μL of sample with the MB into Eppendorf tubes, washings, incubation with TMB for 30 min, acid treatment, and spectrophotometric detection. When run in PBS−BSA, the assay successfully measured MPO concentrations between 180 and 2000 ng mL−1, with a LOD of 79 ng mL−1 and LOQ of 180 ng mL−1. Finally, the potential matrix effect of real serum samples on assay performance was evaluated. With this purpose, three different sample matrixes (PBS−BSA, blood serum obtained from a healthy individual diluted with PBS 1:100, and the same serum diluted 1:50) were spiked with MPO at similar concentration levels, and three calibration curves were obtained in parallel (Figure 2d). As expected, the signals generated for each MPO final concentration were inversely proportional to the sample complexity. However, at MPO concentrations lower than 12.5 μg mL−1 in prediluted samples, signal-to-noise ratios obtained in 1:50 serum were at least 10% higher than those in 1:100 serum. Hence, real serum samples were diluted 1:50 in subsequent experiments because of the better performance in low MPO concentrations and the low LOD required. Electrochemical Measurements. The optimized magneto immunoassay was next transferred to an electrochemical biosensing format. MPO immunocapture was carried out as described above, and following washing, MBs were layered onto the surface on an electrode. The electrode had been placed in the modular holder described in Design and Fabrication of a Modular Holder (Figure 1). Electrochemical Detection of MPO Activity. As before, detection took advantage of the endogenous peroxidase activity of MPO using TMB/H2O2. Since peroxidase activity results in TMB oxidation, electrochemical detection was performed on the basis of the reduction of the oxidized TMB at the electrode surface. The electroanalytical response of five different types of electrodes (Table 1) was studied in parallel by detecting chronoamperometrically MPO serial dilutions. The optimal electrode for MPO determination was then selected on the basis of detection sensitivity, reproducibility, linear range, and LOD (Figure 3). When comparing electrodes 1−3 to electrodes 4 and 5, assay performance improved, in terms of higher slope and lower LOD, with the electrode area (Figure 3). The most significant exception was electrode 3, which in spite of its size, displayed the highest sensitivity, the lowest LOD, and the widest assay linear range. Since the major difference between electrodes 2 and 3 was the reference electrode (thin-film Pt pseudoreference versus thick-film Ag/AgCl, respectively), the divergence in behavior was presumably caused by reference potential shift over the measurement in the former case. Unexpectedly, while the utilization of the CNT-modified electrode 5 produced a better LOD compared to electrode 4, a notably narrower linear range was also obtained. In accordance with these results, electrode 3 was used in the rest of the experiments. MPO Detection Using the Electrochemical Magneto Immunoassay. In the next step, MPO electrochemical detection was undertaken by combining the optimized magneto immunoassay and cronoamperometric detection of the enzyme

Figure 3. Electrochemical detection of MPO in solution. (a) Calibration plots of current versus concentration of MPO. (Inset) Examples of the chronoamperograms registered for 300 ng mL−1 MPO using the five types of electrodes. (b) Calibration parameters obtained for the five types of electrodes that were tested. LOD = Sb + 3σ, σ = black standard deviation (n = 6), and Sb = averaged blank signal (n = 6).

activity at the 3D holder using the electrode type 3. With this aim, MPO was serially diluted in 1% PBS−BSA and, before incubation with the MBs, was additionally diluted 1:50 with PBS to simulate the conditions in which the assay should be performed in real samples. Following immunocapture and washing, MBs were layered onto the electrode surface, were covered with 50 μL of PBS, and current was registered at 0 V until stabilization was reached (approximately 150 s). The measurement was then paused, PBS was substituted with TMB solution, and measurement was resumed for other 300 s (Figure 4a). Under these conditions, the response was linear for starting MPO concentrations between 45 and 3000 ng mL−1 (0.9−60 ng mL−1 after dilution), with a sensitivity of −0.052 ± 0.002 nA mL ng−1 (RSDn = 3 of 1.5%, r2 = 0.998, 95% confidence). The LOD of MPO was 20 ng mL−1 in prediluted samples, equivalent to 0.4 ng mL−1 after dilution. Other authors have previously reported immunosensors displaying slightly wider linear ranges, LODs 1 order of magnitude lower and assay times of 15−20 min.16−19 However, those four examples used nanostructured electrodes based on long, complex, and tedious production procedures that would be difficult to transfer for their real application in the diagnostics field. With respect to the MPO concentration commonly found in human plasma of healthy and CVD patients (

Chronoamperometric Magneto Immunosensor for Myeloperoxidase

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