Article pubs.acs.org/ac
Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Micromotor-Assisted Human Serum Glucose Biosensing Lei Kong,†,‡ Nasuha Rohaizad,†,§ Muhammad Zafir Mohamad Nasir,† Jianguo Guan,*,‡ and Martin Pumera*,†,∥,⊥,#
Anal. Chem. Downloaded from pubs.acs.org by UNIV AUTONOMA DE COAHUILA on 04/16/19. For personal use only.
†
Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371, Singapore ‡ State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, People’s Republic of China § NTU Institute for Health Technologies, Interdisciplinary Graduate School, Nanyang Technological University, Singapore 637553, Singapore ∥ Center of Advanced Functional Nanorobots, Department of Inorganic Chemistry, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic ⊥ Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea # Future Energy and Innovation Laboratory, Central European Institute of Technology, Brno University of Technology, Purkyňova 656/123, Brno CZ-616 00, Czech Republic S Supporting Information *
ABSTRACT: Artificial self-propelled micromachines have shown great promise in biomedical sciences. In this work, we use Mg/Pt Janus micromotors with self-rejuvenating surfaces to enhance the electrochemical sensing performance and sensitivity toward glucose in human serum. The detection of glucose is based on the glucose oxidase enzyme and ferrocenemethanol shuttle system, where mass transfer was dramatically enhanced by the rapid motion of Mg/Pt Janus micromotors. The obtained chronoamperometric data show that Mg/Pt Janus micromotors play a synergistic role in enhancing the current response at millimolar concentrations of glucose in human serum. The current signals increased with the corresponding increase in amount of micromotors introduced. Furthermore, a linear relationship between current signal and glucose concentration was established, while the limit of detection improved when mobile Mg/Pt Janus micromachines were used. Glucose detection enhanced by micromachines may pave the way for their future applications in biomedicine and medical diagnostic devices.
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the catalytic surfaces. These factors greatly limit the applications of micromotors in human blood samples.19−22 Mg-based micromotors have displayed vigorous hydrogen gas bubble generation coupled with fast motion in simulated body fluid (SBF) and blood plasma by a bubble recoil mechanism without any addition of external toxic fuels or surfactants.4 Additionally, Mg dissolves in the plasma, exposing a fresh surface, which enables the reaction to progress continuously. These micromotors also exhibit excellent hemocompatibility and have potential applications in vivo.23−25 Furthermore, the increased fluid transport by the motion and bubble release of Mg-based micromotors was found to be helpful in promoting the efficacy for pollutant degradation and chemical detection.26−28 Thus, Mg-based micromotors offer an alternative strategy for fabricating
ynthetic micro-/nanomotors have attracted considerable attention for potential applications in environmental remediation,1−3 drug delivery,4,5 cell separation,6 and sensing.7−9 In the context of sensing, the enhanced mass transfer generated from the autonomous motion of micromotors would greatly improve the detection sensitivities of target molecules.7,10 As such, biomolecules, ions and pollutants can be effectively detected with the aid of micromotors in the sample solution.11−14 One such potential application would be the detection of blood glucose in humans. The sensitive and rapid detection of blood glucose is a pressing need, given the widespread global increase in diabetic individuals; thereby the accurate detection of sugar in blood has become more crucial.15 Motion-enhanced diffusion and electrogenerated chemiluminescence with enzyme-modified motors have been shown to provide novel and enhanced methods for glucose sensing.16−18 However, these developed catalytic motors have complex components as well as hinder both motion in highviscosity solution and the adsorption of proteins or ions onto © XXXX American Chemical Society
Received: November 27, 2018 Accepted: April 3, 2019
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DOI: 10.1021/acs.analchem.8b05464 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Motion study experiments of Mg/Pt Janus micromotors in the absence of glucose were performed by adding 4.62 μL micromotors suspended in PBS-FcMeOH into 30 μL of an HS/PBS-FcMeOH solution. For motion studies at variable glucose concentrations, 7.5, 15, 37.5, 75, and 112.5 mM glucose solutions were first prepared in PBS-FcMeOH. Subsequently, 4.62 μL of the respective glucose mixtures, in the presence of Mg/Pt Janus micromotors, was added to 30 μL of an HS/PBS-FcMeOH solution, yielding final glucose concentrations of 1, 2, 5, 10, and 15 mM. Electrochemical Detection of Glucose in Human Serum with Mg/Pt Janus Micromotors. Screen-printed electrodes (SPEs) with a three-electrode system were used (Figure S1). The working electrode on SPE was initially modified with glucose oxidase (GOx) and glutaraldehyde (GTA) solution before measurements. First, 3 μL of GOx (0.1 g/mL in ultrapure water) was deposited onto the working electrode and dried under ambient conditions. Subsequently, 3 μL of GTA (0.25% in ultrapure water) was drop-casted and dried in the oven at 35 °C for 30 min. Cyclic voltammetry (CV) measurements for variable glucose concentrations were performed between −0.3 and 0.7 V at a scan rate of 100 mV/s. Each experiment was performed twice on the same SPE. The first scan was carried out with 30 μL of HS/PBS-FcMeOH solution on an SPE. Subsequently, the second scan was performed on the same SPE after injecting 4.62 μL of a glucose/PBS-FcMeOH solution of the desired concentration. Chronoamperometry was performed at a potential of 0.2 V for 400 s. Similarly, each chronoamperometric experiment was performed twice on the same SPE. The first scan was performed with 30 μL of the HS/PBS-FcMeOH solution previously prepared. Subsequently, on the same SPE, 4.62 μL of micromotor/glucose/PBS-FcMeOH suspensions of the desired micromotors and glucose concentrations was injected during the second scan. The chronoamperometric measurements performed with micromotor/PBS-FcMeOH, Mg/PBSFcMeOH, and Pt/PBS-FcMeOH suspensions followed similar procedures. Equipment. Sputtering with Pt was carried out with a JEOL JFC-1600 Auto Fine Coater. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) were carried out by using a JEOL-7600F semi-in-lens field-emission SEM coupled to an Oxford EDX instrument. Optical microscope videos and images were captured with a Nikon Eclipse Ti-E microscope. Cyclic voltammetry and chronoamperometry were performed at room temperature using an Autolab PGSTAT 101 electrochemical analyzer (Eco Chemie, Utrecht, The Netherlands) connected to a computer and controlled by NOVA Version 1.10 software (Eco Chemie). Screen-printed electrodes (SPEs) with a threeelectrode system (Zensor TE100, Taiwan) were used for all experiments.
biocompatible micromotors which can be implemented in biological and medical applications.29 Herein, we aim to improve the electrochemical detection capabilities of glucose in human serum assisted by Mg/Pt Janus micromotors without the need for additional toxic fuels or surfactants. Electrochemical sensing has the advantages of being rapid, sensitive, selective, and low cost in detecting pollutants and biomolecules.30 Extremely low concentrations of target molecules in solutions can be measured with high sensitivity and selectivity through changes in the current signals generated. It has been demonstrated that the immobilization of glucose oxidase (GOx) enzyme onto the electrode surface provides a highly selective method for blood glucose sensing.15,31 Therefore, a second-generation glucose biosensor system was employed which involved the enzymatic oxidation of glucose by GOx and the electrochemical detection of a mediator,32 ferrocenemethanol (FcMeOH). In addition, a screen-printed electrode (SPE) was used, which allowed for the miniaturization of the experimental setup and minimized the amount of sample required.33 The chronoamperometric data show that Mg/Pt Janus micromotors play a synergistic role in enhancing the detection current at millimolar concentrations of glucose in human serum by enhancing the mass transfer in the solution. The current signal increased with an increase in the amount of micromotors. Furthermore, a linear relationship between the current signal and glucose concentration was established and the limit of detection (LOD) was improved with the optimal concentration of Mg/ Pt Janus micromotors present. This opens up the possibility for the fabrication of on-site, point-of-care diagnostic devices with the incorporation of autonomous moving micromotors for rapid sample detection.34
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EXPERIMENTAL SECTION Materials. Mg microspheres with an average diameter of ∼20 μm were purchased from Tangshan WeiHao Magnesium Powder Co. Platinum (powder, ≤10 μm), acetone, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen phosphate, human serum, glucose oxidase from Aspergillus niger (EC 1.1.3.4, type X-S), glutaraldehyde solution (70% in water), and D-(+)-glucose (≥99.5%) were purchased from Sigma-Aldrich, Singapore. Ferrocenemethanol (>95%) was purchased from Tokyo Chemical Industry. Ultrapure water with a resistivity of 18.2 MΩ cm, purified in a Milli-Q system (Millipore, MA, USA), was used for the preparation for all aqueous solutions. Fabrication of Mg/Pt Janus Micromotors. Mg microspheres were washed with acetone by sonication for 10 min before use. Subsequently, 0.3 mL of Mg in an acetone suspension (5 mg/mL) was dropped onto 22 × 22 mm glass slides and dried at room temperature. Mg microspheres were partially covered with Pt by ion sputtering at a current of 40 mA for 200 s. Finally, Mg/Pt Janus microparticles were collected by sonicating the glass slides in acetone and drying in the oven at 35 °C for 1 h. Operation of Micromotors. Phosphate buffer solution (PBS, pH 7.2) was prepared with potassium chloride, potassium dihydrogen phosphate, and sodium hydrogen phosphate dissolved in ultrapure water. A 2 mM solution of ferrocenemethanol (FcMeOH) in PBS was prepared (PBSFcMeOH). The running solution for micromotors was prepared with human serum (HS) and PBS-FcMeOH solution (HS/PBS-FcMeOH, volume ratio 1:3).
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RESULTS AND DISCUSSION The mechanism of Mg/Pt Janus micromotor assisted electrochemical glucose sensing in human serum (HS) is shown in Scheme 1. The working electrode of the screen-printed electrode (SPE) was first modified with glucose oxidase (GOx), followed by the deposition of glutaraldehyde (GTA). The system follows a second-generation glucose biosensor principle where a mediator, FcMeOH, was introduced to facilitate the heterogeneous electron transfer more efficiently B
DOI: 10.1021/acs.analchem.8b05464 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry Scheme 1. Schematic Representation of Mg/Pt Janus Micromotor Assisted Glucose Biosensing in Human Serum Using SPEa
a
The increase in signal output is attributed to the enhanced mass transfer generated by the micromotor motion and bubbling.
Figure 1. (a) SEM image, (b) EDX spectrum, and (c, d) elemental mappings of fabricated Mg/Pt Janus micromotors.
(eq 2) upon the enzymatic breakdown of glucose by GOx (eq 1).32 The current signal was generated by the electrochemical interconversion of FcMeOH to Fc+MeOH (eq 3), which correlated with the glucose concentration in the solution. The proposed Mg/Pt Janus micromotors could move spontaneously in human serum by a bubble recoil mechanism without any external additions, as has been previously proven.4 Additionally, the micromotors exhibit fast motion and vigorous bubbling in the solution, which caused enhanced fluid transport.27,28 The enhanced motion was postulated to increase the flow of molecules and ions, which would accelerate the oxidation of glucose and FcMeOH on the electrode surface, thus improving detection signals.35
The Mg(OH)2 passivation layer produced can be effectively removed from pit corrosion by Cl− ions present in human serum and the buffering effects of blood plasma.4 This would expose a fresh Mg surface which facilitates the Mg−H2O reaction to generate hydrogen bubbles continuously and uninterruptedly. We first studied the motion of Mg/Pt Janus micromotors with human serum (HS) dissolved in PBSFcMeOH (HS/PBS-FcMeOH, volume ratio 1:3). The videos were captured in the presence and absence of glucose with micromotors suspended in HS/PBS-FcMeOH solution (see details in the Experimental Section). The trajectory of Mg/Pt Janus micromotors in HS/PBS-FcMeOH solution was tracked for 3 s in the absence of glucose (Figure 2a). Many bubbles were continuously generated, and the micromotor was
glucose + FAD‐GOx + 1/2O2 → gluconic acid + FADH 2‐GOx + H 2O2
(1)
FADH 2‐GOx + 2Fc+MeOH → FAD‐GOx + 2FcMeOH + 2H+ 2FcMeOH → 2Fc+MeOH + 2e−
(2) (3)
The Mg/Pt Janus micromotors were fabricated by sputtering Mg particles with Pt (see details in the Experimental Section) and characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). A high-magnification SEM image shows a typical Janus particle (Figure 1a), indicating its spherical shape with a diameter of about 30 μm. The Pt coating on the right side of the Mg surface shows the asymmetric structure of the particle. To confirm and further investigate the Janus structure, an EDX spectrum and elemental mapping were carried out. The EDX spectrum (Figure 1b) indicates that the particle consists of mainly the elements Mg and Pt, while the Si signal comes from the silicon substrate. At the same time, EDX mappings (Figure 1c,d) confirm that Mg and Pt are concentrated on the opposite sides of the Janus particle, respectively, which is consistent with the asymmetric morphology in the SEM image. From previous studies,4,28 Mg-based micromotors move in blood plasma or human serum by a bubble recoil mechanism as a result of hydrogen bubble production on the basis of eq 4: Mg + 2H 2O → Mg(OH)2 + H 2
Figure 2. Trajectories of Mg/Pt Janus micromotors in solution containing HS/PBS-FcMeOH in the (a) absence and (b) presence of 1 mM glucose for a duration of 3 s. (c) Average velocities and lifetimes of Mg/Pt Janus micromotors, which were obtained from statistical measurements of at least 30 different micromotors from the aforementioned conditions, respectively.
(4) C
DOI: 10.1021/acs.analchem.8b05464 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry observed to move at a very fast velocity (Video S1). We next investigated the motion of the fabricated micromotor in HS/ PBS-FcMeOH solution with 1 mM glucose, as shown in Figure 2b. It was observed that the micromotor did not display a noticeable decrease in performance, which suggests that the movement is not affected by the addition of glucose. The fabricated Mg/Pt Janus micromotors showed excellent movement in HS/PBS-FcMeOH solution without the need for additional fuels or surfactants. The average velocities and lifetimes were calculated with at least 30 different micromotors under each set of experimental conditions (Figure 2c). The average velocities in the presence and absence of 1 mM glucose were rather similar at 80.6 ± 28.5 and 86.8 ± 31.1 μm/s, respectively. The lifetimes of the Janus micromotors were also consistent at 53 ± 27 and 55 ± 29 s, respectively. Additionally, the fabricated Mg/Pt Janus particles were observed to continue producing bubbles for several minutes despite no motion being observed. As such, the continued bubbling effect would provide additional flow in the solution despite the lack of movement of the micromotors.27 Having ascertained the motion of the fabricated micromotors in human serum solution, we next set forth to analyze the performance of the proposed glucose biosensor system. The fabrication of the glucose biosensor is described in the Experimental Section and a digital photograph of the experimental setup is shown in Figure S1. Cyclic voltammetry was carried out between −0.3 and 0.7 V at a scan rate of 100 mV/s. Two scans were performed on the same SPE for each experiment. The first voltammetric scan was performed on 30 μL of an HS/PBS-FcMeOH solution (volume ratio 1:3), and the second scan was performed after injecting 4.62 μL of glucose solutions of different concentrations into HS/PBSFcMeOH on the same SPE (see details in the Experimental Section). Subsequently, the differences in anodic peak heights, corresponding to the current difference (ΔI) between both scans, were calculated to quantify the biosensor response at different glucose concentrations. Figure 3 shows the cyclic voltammograms obtained at different glucose concentrations, where an oxidation peak was observed at ∼0.2 V. The results show that ΔI (at a potential of 0.2 V) increased with a
corresponding increase in glucose concentration varying from 0.5 to 1, 2, and 5 mM. Henceforth, the proposed glucose biosensor system displayed a positive correlation with the corresponding increase in glucose solution introduced. Subsequently, we investigated the effect of introducing Mg/ Pt Janus micromotors into the biosensor system. Chronoamperometry was performed at a potential of 0.2 V on an HS/ PBS-FcMeOH solution in the presence of 1 mM glucose solution at different concentrations of Mg/Pt Janus micromotors. An applied potential of 0.2 V was selected, as it corresponded to the oxidation peak observed for the glucose concentrations analyzed in Figure 3. Similarly, the chronoamperometry experiments were performed on the same SPE for two scans. The first scan was performed on 30 μL an HS/PBSFcMeOH solution, which served as the background signal. The second scan was performed on the same SPE. The background current was allowed to stabilize for 100 s before injecting 4.62 μL of a micromotor/glucose/PBS-FcMeOH suspension (see details in the Experimental Section).32 Figure S2a−d shows the chronoamperometric measurements obtained. It was noted that, in the absence of a micromotor and glucose mixture, the current signal showed a gradual decrease with time (blue lines). However, upon the injection of micromotors at time 100 s during the second scan (red lines), the current signals increased sharply owing to the disturbance near the surface of the electrode and the localized high glucose concentration.36 The current signal decreased quickly and eventually stabilized. On comparison of the chronoamperometric measurements between the first and second scans, the current signals generated from the second scans showed higher values due to the presence of additional glucose molecules. The differences in current signals (ΔI) at a time of 300 s between two scans were measured to analyze the effect of micromotor concentration on the response of the biosensor system and calculated with eq 5: ΔI = I2nd − I1st
(5)
I1st represents the measured current in the first scan (blue line), and I2nd represents the measured current in the second scan (red line) at a time of 300 s. A ΔI value of 0.25 μA was obtained in the control experiment in the absence of Mg/Pt Janus micromotors (Figure S2e). The ΔI values obtained gradually increased with a corresponding increase in Mg/Pt Janus micromotor concentration, which suggests that the moving fabricated micromotors increased the performance of the glucose biosensor. To further understand the contributory effect of the fabricated micromotors on the enhanced signals obtained, additional control experiments were performed. Figure 4a illustrates the resultant chronoamperograms obtained after deducting the background signal, as calculated with eq 5. The experiment was initially performed with the injection of a PBSFcMeOH solution, which served as the background and reference current signal (ΔIR). Subsequently, Mg, Pt, and Mg/ Pt suspensions were injected to analyze the differences in performances of the different suspensions. From the inset in Figure 4a, it was observed that the current signal obtained with Mg/Pt Janus micromotors (blue line) was significantly higher in comparison to the other current signals obtained. The ΔI values at 300 s were calculated and plotted (Figure 4b) to provide a better representation of the different electrochemical performances for the different conditions analyzed. From Figure 4b, the addition of PBS-FcMeOH solution showed the
Figure 3. Cyclic voltammograms of HS/PBS-FcMeOH at glucose concentrations of (a) 0.5 mM, (b) 1 mM, (c) 2 mM, and (d) 5 mM (scan rate 100 mV/s). D
DOI: 10.1021/acs.analchem.8b05464 Anal. Chem. XXXX, XXX, XXX−XXX
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We then investigated the effect of micromotor concentration on the performance of the biosensor system. Chronoamperometry was performed in HS/PBS-FcMeOH solutions containing 1 mM glucose at different concentrations of Mg/ Pt Janus micromotors (Figure 5a). From the chronoampero-
Figure 4. (a) Chronoamperometric measurements of HS/PBSFcMeOH with different additions. The inset shows the values obtained at a time of 300 s. (b) Comparison of current differences (ΔI) calculated at 300 s using eq 5.
only negative ΔIR value of −0.19 μA. This could be attributed to the absence of glucose molecules during the second scan, which resulted in a lower current signal obtained (the purchased human serum contains a concentration of glucose in it). The ΔI obtained upon addition of Pt particles is 0.21 μA, which is almost the same as that obtained with glucose solution in the absence of micromotors (0.25 μA). This would suggest that Pt does not exert any significant interference on the measurements. However, the ΔI value obtained with Mg particles was significantly higher (0.44 μA). It was initially postulated that the Mg particle solution would have current signals similar to those of glucose solution due to the low reaction rate between Mg particles and water (Video S2).37 However, from previous studies,38 Mg was found to react with Fc+MeOH due to the strong reducibility. Hence, more FcMeOH molecules would be produced, which would lead to the increment of oxidation current from the interconversion between FcMeOH and Fc+MeOH. In PBS-FcMeOH solution without glucose, it was observed that both Mg and Pt particles displayed negative ΔI values while only Mg/Pt Janus micromotors showed positive ΔI values (Figure S3). A comparison with ΔIR (−0.19 μA) indicates that the Pt particles do not influence the measurement, and the increment of ΔI from Mg/Pt Janus micromotors and Mg particles can be ascribed to the Mg-Fc+MeOH reaction.20 Therefore, the significantly higher ΔI value obtained from the addition of Mg/Pt Janus micromotors is the synergistic effect between the enhanced mass transfer by the fast motion of micromotors and the reaction between Mg and Fc+MeOH. Accordingly, a revised ΔI′ value can be obtained by deducting the ΔI value caused by the Mg-Fc+MeOH reaction on the basis of eq 6 ΔI ′ = ΔIM&G − (ΔIM − ΔIR )
Figure 5. (a) Chronoamperometric measurements in HS/PBSFcMeOH with 1 mM glucose solution at different concentrations of Mg/Pt Janus micromotors injected. The inset shows the current signals obtained at 300 s. (b) Comparison of current differences (ΔI) calculated at 300 s with eq 5 at different concentrations of Mg/Pt Janus micromotors.
metric measurements obtained, it was noted that the ΔI values generally increased with a corresponding increase in Mg/Pt Janus micromotor concentration. This is in accordance with the increase in bubbling effects, which contributed to the increased motion in the solution with more micromotors present per unit volume. As such, there would be a corresponding increase in enzymatic reactions and electrode reactions occurring because of the the enhanced fluid flow in the solution, resulting in higher electrochemical currents detected.35 However, the current signal plateaued at a micromotor concentration of 10 mg/mL and decreased beyond that concentration. The basis of the propulsion of the fabricated micromotors stems from the reaction between Mg and water, producing hydrogen gas and Mg(OH)2, a base (eq 4). This would suggest that, at very high micromotor concentration, a significantly higher quantity of base would be produced, which could increase the pH of the solution and might affect the performance of the enzyme, glucose oxidase. The pH values of the micromotor and glucose mixtures at different micromotor concentrations were measured after 5 min of reaction, and the results obtained showed pH values below 9 at all micromotor concentrations. These findings indicated no significant influence of pH on the activity of glucose oxidase in the biosensor system.39 Upon closer investigation of the motion behavior of the Mg/Pt Janus micromotors, it was observed that some particles did not move and settled at the bottom of the glass slide (Figure S4), probably a result of defects in the fabrication process. Bubbles produced from the Mg/Pt Janus particles could also adhere on the enzyme/ electrode surface. These factors would passivate and reduce the available exposed surface area for enzymatic catalysis and electrode reaction. As such, at higher micromotor concentrations, this effect would become more apparent, which could result in the drop in current signals observed in Figure 5. The ΔI values obtained for Mg/Pt micromotors at concentrations of 2.5 and 5 mg/mL in the absence of glucose were also measured (Figure S4f), which were 0.56 and 1.3 μA, respectively. The revised ΔI′ values were 0.65 and 0.42 μA, indicating that Mg/Pt Janus micromotor concentrations of 2.5 and 5 mg/mL improved the detection signal in the presence of
(6)
where ΔIM&G is the measured current for the micromotor/ glucose mixture and ΔIM represents the measured current for the micromotor solution in the absence of glucose. From eq 6, the revised ΔI′ values for micromotor/glucose and Mg/ glucose mixtures should be 0.36 and 0.26 μA, respectively. This would indicate that the introduction of Mg/Pt Janus micromotors led to an increment of 0.11 μA in the current signal for 1 mM glucose detection, which is attributed to the enhanced mass transfer in the solution by the fast motion and bubbling of micromotors. However, the addition of Mg particles did not result in much improvement due to their lower reaction rate with the medium, which resulted in significantly lesser bubbling. E
DOI: 10.1021/acs.analchem.8b05464 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry 1 mM glucose (0.25 μA). Nonetheless, the addition of 5 mg/ mL of Mg/Pt Janus micromotors would be less efficient due to the higher possibility of loss of electroactive sites on the enzyme/electrode. The effect of varying glucose concentration was next studied at micromotor concentrations of 1 and 2.5 mg/mL. The visual motions of Mg/Pt Janus micromotors in HS/PBS-FcMeOH in the presence of 2, 5, 10, and 15 mM glucose solutions were first studied (Figure S5 and Video S3). The fabricated micromotors were observed to generally display similar velocities and lifetimes in solutions containing 1 mM glucose concentration (Figure 2). This would suggest that the motion of micromotors is not significantly limited or affected by the amount of glucose molecules present in the solution and thus the micromotors continue to exhibit similar performances. From the chronoamperometric measurements obtained (Figures 6a−c), the electrochemical current signals increased
Janus micromotors, respectively. At the same time, the limit of detection (LOD) and limit of quantification (LOQ) values were calculated on the basis of the IUPAC approach.40 In general, the LOD was obtained by multiplying the quotient of the standard deviation of the chronoamperometric current at the lowest glucose concentration and gradient of the calibration graph by 3. Conversely, the LOQ was obtained by multiplying 10 to the same quotient previously described. In the absence of micromotors, the LOD and LOQ values obtained were 54.1 and 180.3 μM, respectively. The introduction of Mg/Pt Janus micromotors induced a change in the LOD and LOQ values obtained. In the presence of 1 mg/mL of micromotors, the LOD and LOQ values obtained were 33.2 and 110.7 μM, respectively. An increase in the concentration of micromotors present to 2.5 mg/mL yielded LOD and LOQ values of 98.4 and 328 μM, respectively. The results obtained would suggest that the introduction of 1 mg/ mL of micromotors could aid in improving the sensitivity of the biosensor system in relation to the lower LOD value obtained. The interferences induced at higher micromotor concentration, as previously discussed above, could have led to larger standard deviations obtained for the currents measured. The results obtained affirm that the detection signals were improved as a consequence of the enhanced mass transfer caused by the motion and bubbling effect of the fabricated Mg/Pt Janus micromotors. Increasing the concentration of Mg/Pt Janus micromotors would result in more micromotors available per unit volume, thus enhancing fluid flow throughout the solution. Furthermore, increasing the glucose concentration from 1 to 15 mM at a constant micromotor concentration showed a positive correlation and good linearity. This could be attributed to more glucose molecules being involved in the reaction due to the efficient mass transfer in the solution.
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CONCLUSION In conclusion, we have demonstrated the improved electrochemical detection of glucose in human serum solution assisted by Mg/Pt Janus micromotors. Mg/Pt Janus micromotors showed excellent autonomous motion in human serum solutions containing different concentrations of glucose without additional toxic fuels or surfactants. The enhancement in signal current detected for glucose in human serum with Mg/Pt Janus micromotors present is attributed to the enhanced mass transfer in the solution induced by the fast motion of micromotors. The proposed system displayed good correlation with an obvious increment in current signals detected at increasing micromotor and glucose concentrations.
Figure 6. Chronoamperometric measurements with different concentrations of glucose in the (a) absence and presence of (b) 1 mg/mL and (c) 2.5 mg/mL of Mg/Pt Janus micromotors. (d−f) Corresponding calibration plots between glucose concentration and electrochemical current signal (ΔI′) calculated with eq 6 at a time of 300 s.
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ASSOCIATED CONTENT
S Supporting Information *
with a corresponding increase in glucose concentration (from 1 to 15 mM) at both concentrations of Mg/Pt Janus micromotors analyzed (1 and 2.5 mg/mL). The ΔI′ values were obtained and tabulated (Table S1), which provided a greater illustration of the direct relationship between the glucose and micromotor concentrations. Calibration plots between electrochemical current signal (ΔI′) and glucose concentrations at different micromotor concentrations were plotted, as illustrated in Figures 6d−f. Figure 6d represents the calibration plot obtained in the absence of micromotors, where a linear regression (r) of 0.975 was obtained. Calibration plots represented by Figures 6e,f were obtained in the presence of 1 mg/mL (r = 0.981) and 2.5 mg/mL (r = 0.985) of Mg/Pt
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b05464. Digital photograph, chronoamperometry data, optical microscope images, micromotor trajectories, and calculated current signals (PDF) Motion of Mg/Pt Janus micromotors in a running solution containing HS/PBS-FcMeOH or HS/PBSFcMeOH with 1 mM glucose (AVI) Mg microparticles in an HS/PBS-FcMeOH solution (AVI) F
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Motion of Mg/Pt Janus micromotors in a running solution containing HS/PBS-FcMeOH with 2, 5, 10, or 15 mM glucose (AVI)
AUTHOR INFORMATION
Corresponding Authors
*E-mail for J.G.:
[email protected]. *E-mail for M.P.:
[email protected]. ORCID
Jianguo Guan: 0000-0002-2223-4524 Martin Pumera: 0000-0001-5846-2951 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the project Advanced Functional Nanorobots (reg. CZ.02.1.01/0.0/0.0/15_003/0000444 financed by the EFRR). The authors acknowledge A*STAR Grant SERC A1783c0005 (Singapore). L.K. acknowledges the Scholarship Fund from the China Scholarship Council (CSC).
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DOI: 10.1021/acs.analchem.8b05464 Anal. Chem. XXXX, XXX, XXX−XXX
