Anti-Biofouling Isoporous Silica-Micelle Membrane Enabling Drug

Letter pubs.acs.org/ac

Anti-Biofouling Isoporous Silica-Micelle Membrane Enabling Drug Detection in Human Whole Blood Qinqin Sun, Fei Yan, Lina Yao, and Bin Su* Institute of Analytical Chemistry, Department of Chemistry, Zhejiang University, Hangzhou 310058, China S Supporting Information *

ABSTRACT: Direct electrochemical detection in human whole blood remains challenging due to electrode surface fouling and passivation by abundant biological substances. In this work we report that the isoporous silica-micelle membranes (designated as iSMM) can effectively function as antibiofouling layer for electrochemical detection of drug molecules in human whole blood without pretreatment. The iSMM possesses molecular sieving capacity, charge/lipophilicity selectivity, and preconcentration ability. Only small and neutral/lipophilic analytes can permeate the iSMM, be concentrated, and subsequently be detected at the underlying electrode. It is however impermeable to big sized substances and those small but charged and hydrophilic. We first investigated the molecular permeability of iSMM by electrochemical impedance spectroscopy (EIS) and then demonstrated its application in the quantitative determination of chloramphenicol (CAP) in the unprocessed human whole blood. The analytical sensitivity and long-term stability of iSMM based electrochemical sensors are apparently better than bare electrodes.

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caused by certain proteins. However, as pointed out by the authors, the nanopores with a size of ∼5−50 nm cannot prevent the surface from the nonspecific adsorption of small redox-active protein, i.e., cytochrome c(with a size of 2.6 nm × 3.2 nm × 3.3 nm).23 In this work, an ultrathin isoporous silica membrane filled with surfactant micelles, designated as iSMM (as illustrated in Scheme 1), was utilized as the antibiofouling layer on the

irect quantitative detection of small molecules by electrochemical methods in contaminant-ridden clinical samples, especially in human whole blood, is a challenging task for building point-of-care (POC) diagnostic devices.1−3 Blood relevant small molecules, referring to a group of low molecular weight compounds, such as drugs, glucose, adenosine triphosphate, peptide, and metabolites, are widely distributed in blood.2−4 Accurate values of blood drugs play an important role in the evaluation of the effectiveness of drugs and help to monitor the healthy condition of patients. However, an enduring challenge in the elaboration of electrochemical whole blood-compatible devices is the surface biofouling.2,4 Biofouling mainly arises from the nonspecific adsorption of biological substances, i.e., lipids, polysaccharides, cells, and proteins, on the device surface during the measurement in complex biological fluids, and hence adversely affects the analytical performance significantly by hampering the transport of analytes and signal transduction. To circumvent the surface biofouling, a variety of strategies based on chemical/physical modification of the conductive electrodes have been brought forward, involving the use of polyethylene glycol (PEG),5−8 polycarbonate microporous membrane, 9−11 zwitterionic phenyl, 12−16 nanostructure gold,17,18 aluminum oxide nanopores,19 and nanomaterial.20−22 Although the biofouling process can be drastically inhibited by the modification layer, the impeditive transport of redox targets or mediators by layers concurrently comes about. How to design a surface to reduce or eliminate biofouling but simultaneously not passivating the surface activity is of significant importance. Recently, Collinson and co-workers17 have reported that the nanoporous gold layer with specific topography can effectively regulate the surface biofouling © XXXX American Chemical Society

Scheme 1. Isoporous Silica-Micelle Membrane (iSMM) with Anti-Biofouling Capacity, Size, Lipophilicity/Charge Selectivity, and Preconcentration Ability for Electrochemical Detection of Drugs in Human Whole Blood

Received: May 27, 2016 Accepted: August 15, 2016

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DOI: 10.1021/acs.analchem.6b02091 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry electrochemical sensor surface for direct detection of drugs in human whole blood. Its antifouling ability arises from (i) the size sieving capacity of ultrasmall silica mesochannels with a diameter of about 2.3 nm and (ii) the lipophilicity/charge selectivity of micelles with hydrophobic cores formed by the organized assembly of the aliphatic tails of the cetyltrimethylammonium bromide (CTAB). Only small and lipophilic/ neutral analytes can permeate the iSMM and be subsequently detected at the underlying electrode, whereas the permeation of large, charged, and hydrophilic substances are strongly prohibited. Moreover, the micelles can also extract and preconcentrate analytes from aqueous solution via the hydrophobic force to increase the analytical sensitivity. In addition, a high density of regularly and vertically aligned mesochannels and surfactant micelles guarantees a high permeability and a low impeditive effect on mass transport to and redox conversion at the underlying electrode surface. As a proof-of-concept, chloramphenicol (CAP, see its structure in Figure S1) was quantitatively determined as a target of analyte in human whole blood directly without sample pretreatment. As a broad-spectrum antibiotics, CAP is widely used in the treatment of serious infections such as typhoid fever and salmonellosis;24 however, the excessive levels in human blood have adverse side effects on human health such as aplastic anemia, leukemia, and gray baby syndrome.25−27 Various techniques have been employed for the detection of CAP, such as photoinduced chemiluminescence,28 HPLC with UV detection, single MS and tandem MS detection,29 capillary electrophoresis with amperometric detection,30 and surface plasmon resonance.31 Although these methods are accurate and selective, their high cost and time-consuming, complicated sample pretreatment (i.e., filtration, extraction, enrichment) limit their applications. By contrast, electrochemical method offers the advantages of simplicity, low cost, high sensitivity, and relative short analysis time. For instances, Agüi ́ et al.32 have developed a voltammetric method for CAP detection with electrochemically activated carbon fiber microelectrodes; Xiao et al.33 have modified the glassy carbon with a novel composite film, comprising of single-wall carbon nanotubes, gold nanoparticle, and ionic liquid, for the detection of CAP. In addition, nitrogen-doped graphene nanosheets decorated with gold nanoparticles34 and multiwall carbon nanotube-CTAB-poly(diphenylamine) coated electrodes have also prepared for CAP detection in complex samples (such as milk, honey, and eye drops). Although satisfactory analytical results were obtained in these published works (see Table S1), detection CAP in unprocessed complex sample still remain challenging. The iSMM was prepared on the surface of indium tin oxide (ITO) glass by the modified Stöber method,35 thus obtained electrode is designated as iSMM/ITO. As characterized by electron microscopy (see Figure S2), the film consisted of a high density of regularly aligned and vertical silica mesochannels of 2.3 nm in diameter and 111 nm in length, in which cylindrical micelles of CTAB were physically confined. The density of channels and micelles is pretty high, ∼4 × 1012 cm−2. Because of the excellent and selective sieving effects in terms of molecular lipophilicity, size, and charge, this film indeed has attracted extensive concern of electrochemists.36,37 Its antifouling ability and compatibility with real/complex sample analysis have been previously reported.38−40 As shown in Figure 1a, voltammetric data suggest that only neutral/small FcMeOH can permeate the iSMM and access to the underlying ITO surface, whereby being oxidized to generate

Figure 1. (a) Cyclic voltammograms (CVs) obtained at a scan rate of 50 mV s−1 and (b) schematic illustration of transport of different redox probes at the iSMM/ITO electrode, the inset in part a is the CV curve of rutin at bare ITO. (c) Impedance Nyquist plots of different probes at the iSMM/ITO electrode. The inset is enlarged view in the high frequency range. (d) The equivalent circuit describing the electrochemical response of FcMeOH at the iSMM/ITO electrode. Rct, W, and Rs represent the charge-transfer resistance, mass-transport related Warburg element, and uncompensated solution resistance, respectively. Cito/m and Cm/s denote the capacitance of the ITO/micelle and micelle/solution interfaces, respectively. In all cases, Fe(CN)63−(0.5 mM, green line), Ru(NH3)63+ (0.5 mM, blue line), FcMeOH (0.5 mM, red line), and rutin (40 μM, approximately saturated solution, black line) were employed and the supporting electrolyte was 0.1 M KCl.

the corresponding current signal. By contrast, charged/ hydrophilic Fe(CN)63− and Ru(NH3)63+ and big sized rutin were unable to cross the iSMM to reach the ITO surface (as exemplified in Figure 1b), thus the current response was only dominated by the capacitive current over the potential range. In fact, Fe(CN)63− and Ru(NH3)63+ can access the ITO surface after the removal of CTAB micelles from the film (namely, ITO modified by the bare isoporous silica membrane, designated as iSM/ITO), as illustrated in Figure S3. However, the redox response of rutin still cannot be observed at the iSM/ITO electrode, due to the size-exclusion effect. The oxidation/ reduction of rutin was only observed at the bare ITO electrode (the inset in Figure 1a). These voltammetric results indicated the selective permeability of iSMM toward uncharged, hydrophobic, and small sized analytes. Although voltammetry data can quickly reflect the selective permeability of iSMM to different analytes, the Faradaic currents are influenced not only by the film structure but also the reaction kinetics. Therefore, electrochemical impedance spectroscopy (EIS) measurement was also performed. EIS is a powerful tool to probe the interfacial structure and charge transfer changes between electrodes individually.41−43 Figure 1c shows the Nyquist plots of four different redox probes at the iSMM/ITO electrode. For charged/hydrophilic or big sized species, i.e., Fe(CN)63−, Ru(NH3)63+, and rutin, huge semiB

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Analytical Chemistry circles were displayed at high frequencies (the green, blue and black curves in Figure 1c), confirming the charge/lipophilicity/ size exclusion function of micelles. The size of semicircles reflects the charge transfer resistance, namely, Rct. A huge value of Rct can be attributed to the impermeable micelles and the insulating silica. By contrast, a much smaller semicircle was observed for neutral FcMeOH (see inset in Figure 1c), indicating the iSMM permeability to FcMeOH and the electron exchange at the ITO surface was effective. The impedance responses of FcMeOH at a bare ITO and iSM/ITO electrode were also recorded for comparison (see Figure S4). The equivalent circuits for FcMeOH at a bare ITO and iSM/ITO electrode consist of the solution resistance Rs, the parallel combination a capacitor Cito/s (or constant phase element CPE1) with a charge-transfer resistance Rct and mass-transport related Warburg element (or CPE2). By contrast, the circuit for FcMeOH at the iSMM/ITO electrode (as shown in Figure 1d) consists of one more element, namely, the capacitance at the micelle/solution interface (Cm/s), associated with the extraction process of FcMeOH into the micelle phase. The values for each element, obtained by closest fitting the experimental data to the corresponding equivalent circuits, are summarized in Table S2. A significantly smaller Rct for FcMeOH at the iSMM/ITO electrode (namely, 188 Ω), compared to those at a bare ITO and iSM/ITO (namely, 1937 Ω and 466 Ω, respectively), is indicative of a fast electron transfer at the iSMM/ITO electrode. This coincides with the fact that the bare ITO surface exhibits slow and sluggish kinetics for electrochemical reactions of many organic compounds.44 Micelles orderly assembled on the ITO surface may provide well-defined microenvironments to favor molecular redox reactions. The primary goal of this work is to demonstrate that the iSMM can function as the antifouling layer for electrochemical analysis of drug in human whole blood. CAP was chosen as the model analyte. We first studied the electrochemical behavior of 1 ppm of CAP dissolved in 10-times diluted human whole blood at different electrodes by DPV. Before electrochemical measurement, the 0.9 wt % NaCl (10.8 mL) was first bubbled with Ar gas for 20 min to exclude the dissolved oxygen and then human whole blood (1.2 mL) was added. To achieve a sensitive voltammetric determination of CAP, mechanical stirring (400 rpm) for 30 s was employed as the preconcentration method (see details in Figure S5). The inset in Figure 2a shows the three-electrode cell used for direct electrochemical measurement in human whole blood. As demonstrated in Figure 2a, CAP showed a sharp reduction peak at −0.75 V at the iSMM/ITO electrode, whereas the peak was broader at a bare ITO or completely “disappeared” at the iSM/ITO electrode. As previously reported,45 the surface of silica nanochannels is rich in Si−O− groups. In this case, the diffusion of hydrophobic and neutral CAP across the hydrophilic and charged silica nanochannels was not favored, especially at a low concentration, such as 1 ppm. Increasing the concentration of CAP, its reduction could be observed at the iSM/ITO electrode but the reduction peak shifted to a more negative potential (see Figure S6). Moreover, originated from the complexity of the whole blood, many unknown redox moieties can contact with the ITO electrode, leading to the overlapped and “broader” redox peak (see black lines in Figure 2a and Figure S7). Arising from the antifouling and molecular sieving ability of iSMM, the underlying ITO electrode surface is free of nonspecific adsorption, passivation, and disturbance by biointerferences, thus a “sharper” peak was observed. Moreover,

Figure 2. (a) DPVs of 1 ppm of CAP in heparinized human whole blood at a bare ITO (black curve), iSM/ITO (blue curve), and iSMM/ ITO electrode (red curve). The blood sample was diluted by 0.9 wt % NaCl and deoxygenated by Ar gas. Inset: the photograph of the 10fold diluted human whole blood used in the electrochemical analysis. (b) Normalized reduction peak current of 10 ppm of CAP before (0 min) and after the addition of human whole blood at the bare ITO (blue column) and the iSMM/ITO (red column). Error bars represent the standard deviations obtained from three electrodes. (c, d) Topview SEM images of iSMM/ITO (c) and bare ITO (d) taken after incubation with whole blood for 45 min.

thanks to the preconcentration ability of micelles, the magnitude of peak current at the iSMM/ITO electrode is 3.0-fold higher than that at a bare ITO, showing the advantage of the former one in the quantitative analysis. In order to quantitatively evaluate the long-term antibiofouling ability and stability of the iSMM, the reduction peak current of 10 ppm of CAP in human whole blood was continuously recorded by DPV. The normalized current was plotted as a function of time, as shown in Figure 2b. The corresponding response of a bare ITO electrode was also shown for comparison. For both electrodes, the normalized current decreased, but the extent of which was drastically different. As can be seen, the redox signal depleted by ∼75.5% for the bare ITO electrode within 45 min, whereas only ∼21.0% for the iSMM/ITO electrode. A better stability of iSMM/ITO electrode can be attributed to the hybrid membrane structure. To verify the antifouling mechanism of iSMM, whole blood adhesion test was carried out by the SEM imaging (Figure 2c,d). After being in contact with whole blood for 45 min, the surface morphologies of the iSMM/ITO and bare ITO were recorded. We believe the antibiofouling ability of iSMM does not arise from the suppression of interferences adsorption as previously reported for other antifouling layers6−8,12,15,16,20,21 but from the molecular sieving mechanism. The small size and electric inertness of silica mesochannels formed an excellent barrier for large biointerferences, preventing the underlying electrode surface from fouling and passivation. We next expanded the study to evaluate linear sensitivity of the iSMM/ITO in whole blood upon successive increasing the concentration of CAP in the range of 0.1 ppm to 15 ppm. Prior to each electrochemical measurement, mechanical stirring was C

DOI: 10.1021/acs.analchem.6b02091 Anal. Chem. XXXX, XXX, XXX−XXX

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antifouling capacity of different electrodes, where SBlood and SNaCl are the slopes of calibration plots in the whole blood and saline solution, respectively. After calculation, the antifouling capacity of iSMM/ITO and ITO are 0.81 and 0.32, respectively. Apparently, the iSMM/ITO is advantageous in the direct detection of drugs in the human whole blood with an antibiofouling capacity better than bare ITO. Additionally, recovery experiments were also carried out by adding standard solution of CAP with different concentrations to diluted human blood matrixes. Three whole blood samples by different volunteers were employed. The results were compared in Table 2, which proved that the sensor was reliable for CAP detection in human whole blood.

applied for 30 s to extract and concentrate CAP from the whole blood. As depicted in Figure 3a,b, the reduction peak current

Table 2. Recovery of Chloramphenicol in Human Whole Blood Samples Diluted 10 Times by 0.9 wt % NaCl (n = 3)

Figure 3. (a) DPV response of the iSMM/ITO electrode upon successive increase of the concentration of CAP. (b) Calibration plots between the peak current and CAP concentration for the iSMM/ITO (red line) and bare ITO electrode (black line). The parameters for DPV: amplitude (0.05 V), pulse width (0.2 s), and pulse period (0.5 s).

sample no.

added (ppm)

found (ppm)

recovery (%)

RSD (%)

1

0.30 10.00 0.30 10.00 0.30 10.00

0.28 9.73 0.29 10.07 0.30 10.60

93.3 97.3 96.7 100.7 100.0 106.0

1.8 2.9 2.1 3.3 1.1 3.6

2

density at −0.75 V gradually increased with increasing the concentration of CAP, displaying sensitivities of (−1.04 ± 0.02) and (−0.54 ± 0.04) μA cm−2 ppm−1 in the range from 0.1 to 3.7 ppm and from 3.7 to 15 ppm, respectively. The limit of detection (LOD) was calculated as (37.7 ± 0.7) ppb based on a signal-to-noise ratio of 3Sb/m, where Sb is the standard deviation of the blank response and m is the slope of the calibration plot at a low concentration range. Note that CAP peak levels in blood are 10−13 ppm after intravenous administration, and the normal level can be directly detected at the iSMM/ITO electrode. Also, the determination of the excessive levels (50 ppm for gray baby syndrome) can also be realized by dilution of the high concentration solution to the response range.27 Additionally, calibration curves of CAP in whole blood were also obtained at the bare ITO and iSM/ITO electrodes (see Figures S6 and S7), and the analytical results were summarized in Table 1. Apart from much sharper current peaks, a higher

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In summary, we demonstrated the use of iSMM as the antifouling layer for direct electrochemical detection of drugs in human whole blood without pretreatment. The iSMM displayed the molecular sieving capacity, charge/hydrophobic permeability, and preconcentration ability. EIS measurements proved that the micelles with hydrophobic hydrocarbon cores could effectively extract neutral lipophilic molecules from aqueous media and that the extracted redox molecules could undergo a fast electron transfer at the micelle/ITO electrode interface. With these priorities, the detection of CAP in unprocessed human whole blood was accomplished with a low LOD (37.7 ppb), a high sensitivity (−1.04 μA cm−2 ppm−1), a wide dynamic range (0.1−15 ppm), a long-term stability (signal retention by 79.0% in 45 min), and a high recovery. We believe that the iSMM is reliable as the antifouling layer for electrochemical detection in human whole blood as well as many other complex media.

Table 1. Comparison of the Analytical Results of Various Electrodes for the Detection of CAP in Whole Blooda electrode iSMM/ ITO iSM/ITO ITO

sensitivity (μAcm−2 ppm−1)

range (ppm)

−1.04 ± 0.02

0.1−3.7

37.7 ± 0.7

0.994

−0.54 ± 0.04 −0.27 ± 0.06 −0.28 ± 0.08

3.7−15.0 3.0−15.0 0.1−15.0

1623.9 ± 35.0 160.8 ± 4.6

0.996 0.989 0.997

LODb (ppb)

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02091. Experimental details, comparison of different electrochemical methods, electron microscopy graphs, voltammetric behaviors of probes at iSM/ITO, more EIS plots and equivalent circuits of FcMeOH, optimized preconcentration time, and more calibration plots in whole blood and in 0.9 wt % NaCl (PDF)

a Each experiment was done in triplicate. bThe detection limit was obtained using equation, 3Sb/m, where m is the slope of the calibration plot and Sb is the standard deviation of the response which is obtained from 11 reduplicative measurements of the blank diluted whole blood.

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analytical sensitivity in the low concentration range, as well as a low LOD, were also achieved with the iSMM/ITO electrode, showing its advantage in the direct detection in the complex media. In order to compare the antifouling capacity of iSMM/ ITO and bare ITO electrodes, the voltammograms and calibration plots of CAP at the two electrodes were also recorded in the 0.9 wt % NaCl (see Figures S8 and S9 and Table S3). Here we define SBlood/SNaCl to present the

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Homepage: http://mypage.zju.edu. cn/binsu. Notes

The authors declare no competing financial interest. D

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Analytical Chemistry

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grant 21575126, 21335001) and the Zhejiang Provincial Nature Science Foundation (Grant LR14B050001).

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DOI: 10.1021/acs.analchem.6b02091 Anal. Chem. XXXX, XXX, XXX−XXX