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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Paper Electrode-Based Flexible Pressure Sensor for Point-of-Care Immunoassay with Digital Multimeter Zhenzhong Yu,† Yun Tang,‡ Guoneng Cai,† Rongrong Ren,† and Dianping Tang*,† †
Key Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350116, People’s Republic of China ‡ Grinnell College, 1115 Eighth Avenue, Grinnell, Iowa 50112, United States
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S Supporting Information *
ABSTRACT: A novel paper electrode-based flexible pressure sensor modified with multiwalled carbon nanotubes was designed for point-of-care (POC) immunoassay of carcinoembryonic antigen (CEA) with digital multimeter readout. The portable POC testing device consisted of flexible pressure sensor equipped with a paper electrode and connected through syringe tubing to a single-break microplate. The immunoreaction was initially carried out on the microplate with a sandwich-type assay format using platinum nanozyme-labeled secondary antibody for the gas generation. Upon addition of hydrogen peroxide (H2O2), platinum nanozyme (catalase-like mimic) reduced it into hydrogen oxide and oxygen (O2). The overflowing oxygen gas increased the pressure of the multiwalled carbon nanotube-functionalized paper electrode in a homemade pressure-tight system, and the increased pressure could be readily monitored using the paper electrode-based flexible pressure-tight sensor with a digital multimeter readout. The detectable signal mainly derived from the resistance change of pressure sensor because of its deformation with the assistance of the as-generated gas, and the shift in the resistance could be allowed to detect the gas pressure even as low as 80 Pa. Under optimum conditions, pressure sensor-based immunoassay exhibited good resistance responses toward target CEA within a linear range of 0.5−60 ng/mL at a detection limit of 167 pg/mL. Moreover, our strategy provided acceptable reproducibility, precision, high specificity, and good accordance with the commercial CEA ELISA kit for detecting human serum specimens.
T
and suitable signal models have been creatively designed for portable, sensitive, and accurate POC testing, such as temperature,11 color,12 pressure,13 and length.14 Among these common parameters, pressure as a classical physical principle has been widely used to measure the process of chemical and biochemical reactions, because it can be obviously increased in a sealed device by a small quantity of gas from gas-generating reactions.15,16 For POC testing, many gas-generating reactions, including hydrogen,17 carbon dioxide,18 and oxygen,19 are widely used. Ding et al. reported hydrogen-based strategy efficiently catalyzed by CuO/Co3 O 4 nanostructure to sensitively detect the cancer cell.20 Zhu et al. demonstrated a facile method to detect prostate specific antigen using the breakdown of H2O2 accelerated by platinum nanoparticles.21 Compared with other gas-generating reactions, however, the decomposition of H2O2 to O2 is the most common and suitable in POC studies due to its high yield and nontoxic products.22 Theoretically, 100 μL of H2O2 (30%, around 0.9 mmol H2O2) can produce about 0.45 mmol of O2, which is equivalent to 10 mL gas under standard conditions. Such a large volume of gas can cause an obvious increase of pressure over 200 kPa if the gas is confined into a 5.0 mL sealed
o satisfy the increasing demands of healthcare, environmental safety, and food quality, numerous well-developed methods and techniques have been reported for this purpose, such as fluorescence,1,2 electrochemical method,3,4 mass spectrometry,5,6 and so on. However, most require high cost, bulky instruments, and sophisticated operations, thus limiting their widespread applications for resource-limited environments, especially for remote areas and home healthcare settings.7,8 As suggested by the World Health Organization (WHO), the diagnostic techniques offered to developing countries should correspond to the “ASSURED” standard (affordable, sensitive, specific, user-friendly, fast, robust, equipment-free, and deliverable to end users).9 Considering these issues, point-of-care (POC) testing, one of the techniques that can meet these requirements, has drawn considerable attention and developed rapidly in recent decades because of its portable equipment and simple operations.10 Despite many advantages for this detection technology, it is still necessary to develop new protocols and strategies to improve its sensitivity and portability, especially for rapid detection. To successfully develop a new POC testing for immunoassays, there are two significant points for obtaining high sensitivity and minimizing the instruments. One of the key points is to exploit highly effective signal approaches that can be generated efficiently and detected easily. Several excellent © XXXX American Chemical Society
Received: October 10, 2018 Accepted: December 20, 2018 Published: December 20, 2018 A
DOI: 10.1021/acs.analchem.8b04635 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
increases rapidly in the sealed device, where the introduced PtNPs catalyze the decomposition of H2O2 to O2 efficiently. The change of pressure, detected by the flexible pressure sensor and read by a digital multimeter, is used to measure the CEA concentration. The main aim of this work is to develop a simple, portable, and cheap POC testing for ultrasensitive detection of disease in remote areas and daily life. To achieve the sensitive and rapid detection of pressure, the conventional filter paper was chosen for the flexible substrates due to its low cost, easy operation, and rich surface textures. Scanning electron microscopy (SEM; SU8020, Hitachi) was first employed to characterize the morphology and microstructure of filter paper. As exhibited in Figure 1A, there were
container. In addition, many kinds of catalysts, e.g., catalase and platinum nanoparticles (PtNPs), have superior ability to accelerate the decomposition of H2O2, which increases the sensitivity and shortens the detection time. Stimulated by these advantages, the pressure, changed by the breakdown of H2O2, is a suitable and sensitive signal model for POC testing. Another key issue is to employ a simple and sensitive signal readout. As for pressure-based POC testing, a hand-held pressure meter23 and volumetric bar24 have been developed as the pressure readout in the detection systems. Besides these conventional pressure sensors, flexible devices gain much attention owing to their high sensitivity for pressure in recent years. Notably, the microstructure of flexible substrates as the key element in the fabrication process has been investigated in various types.25 As a traditional artifact, the paper is considered to be a potential substrate material due to its low-cost and rich surface textures. Gong et al. used tissue paper coated by gold nanowires to fabricate a sensitive pressure sensor with a wide pressure range from 0.013 to 50 kPa, which could monitor the heartbeat and acoustic vibrations in real-time.26 With such a high sensitivity, it is reasonable to believe that applying a flexible sensor to pressure-based POC testing can obviously improve the detection result. Inspired by the above concerns, a pressure-based POC testing platform is designed for the detection of carcinoembryonic antigen (CEA, as a model analyte) equipped with a paper electrode-based flexible pressure sensor (Scheme 1). A sandwich-type immunocomplex is formed by the CEA, capture antibody, and PtNP-labeled detection antibody in a highbinding microplate (note, PtNPs with ∼40 nm in diameter were used as an example in this case). After adding H2O2 and combining with the flexible pressure sensor, the pressure Scheme 1. (A) Schematic Illustration of Paper ElectrodeBased Flexible Pressure Sensor for Point-of-Care Immunoassay with Digital Multimeter Readout; (B−D) Photographs of (B) Flexible Pressure Sensor, (C) Bendability of Flexible Pressure Sensor, and (D) Flexible Pressure Sensor-Based Immunosensing Device
Figure 1. (A) TEM image of filter paper; (B) photographs and TEM images of filter paper (top two pictures) and MWCNTs-paper (bottom two pictures), respectively; (C) Raman spectra of filter paper and MWCNTs-paper; (D) real-time I−t curve of the pressure sensor at an applied pressure of (a) 0, (b) 1, and (c) 5 kPa; (E) response time (left) and releasing time (right) of the pressure sensor; (F) current response of the pressure sensor to various pressures and its sensitivity (curve S1 and S2).
rich fibrous structures to format a rough microsurface, which was the basis for its sensitive detection of a small pressure change. In order to enhance the conductivity, the filter paper was modified with multiwalled carbon nanotubes (MWCNTs) until the resistance decreased to around 60 kΩ. Typical photographs and SEM images of the filter paper before and after treatment with MWCNTs were shown in Figure 1B, respectively. As seen from the top two photographs of Figure 1B, the original filter paper presents a white color and the cellulose fibers were quite smooth. However, after treatment with MWCNTs solution, it gave a black filter paper. Meanwhile, the surface of cellulose fiber became rough, where the MWCNTs tightly attached to the filter paper to form a consecutive conducting network (bottom two photographs of Figure 1B). In addition, to further verify the B
DOI: 10.1021/acs.analchem.8b04635 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
sensitivity. All in all, these experimental results showed a flexible pressure sensor with high sensitivity, good repeatability, short response time, and a wide pressure range, which would produce excellent performance in the pressure-based POC testing. Before equipping the flexible pressure sensor for the proposed immunoassay as a signal readout, a major concern to be clarified was whether the pressure change caused by the decomposition of H2O2 could be monitored by the pressure sensor. To investigate this point, PtNPs was chosen as the catalyst to accelerate the breakdown of H2O2 due to its good stability and high efficiency. As shown in Figure 2A, the
modification of MWCNTs, Raman spectroscopy (Renishaw) was used to investigate the component. Figure 1C shows the Raman spectra of filter paper and MWCNTs-coated paper. For filter paper, no obvious peak was observed within 1200−1800 cm−1. Meanwhile, the characteristic D band (1352 cm−1) and G band (1584 cm−1) were present in the MWCNTs-coated paper spectrum, which were originated from the defective graphitic structure and C−C stretching vibrations, respectively.27,28 These results revealed that the MWCNTs was successfully assembled on the surface of the filter paper. Such conductive substrate played a significant role in the process of pressure detection and endowed the device with higher sensitivity and shorter response time. To fabricate a highly sensitive pressure-based POC testing, the response of the flexible pressure sensor to pressure change is very crucial. Scheme 1B illustrates the principle of the prepared device, which is based on resistance change between two paper electrodes. When applying pressure on the device, a compressive deformation occurred to enhance the contiguous area between two paper electrodes resulting in more conductive sites. Thus, the current would increase at a fixed applied potential. In the same way, when removing the applied pressure, the device recovered to its initial shape, and the contact area and current were reduced, simultaneously. The unique microstructure of filter paper was the key feature for the device to sensitively monitor pressure due to its great change in the contact area. For elucidating the abilities of the assembled pressure sensor, the device was measured with different pressures on an electrochemical workstation (CHI 850D, Chenhua, China) at an applied voltage of 1.5 V. As shown in Figure 1D, the typical I−t curves were presented with the applied pressures of 0 (curve a), 1 (curve b), and 5 (curve c) kPa, repeatedly. Upon loading a pressure to the device, an obvious current increase was observed and positively related to the increasing pressure. Notably, within several cycles (10 s for each cycle), there was no obvious difference of the current change under the same pressure, which revealed that the pressure sensor was highly repeatable and stable. On the other hand, the response time of the pressure sensor was also an important factor for the detection system. Figure 1E shows the typical curves of the response time and releasing time under 5 kPa, respectively, and both of them were about 0.3 s. Furthermore, the sensitivity was another key point that should be considered, which was defined as29
transmission electron microscopy (TEM; HT-7700, Hitachi) image of prepared PtNPs indicated that the PtNPs had good monodispersity. Figure 2B gives an average size of PtNPs around 40 nm characterized by dynamic light scattering (DLS; Zetasizer Nano, Malvern). Meanwhile, the black PtNPs aqueous solution shown in Figure 2A (inset) was quite stable, resulting in remarkable biocompatibility. Subsequently, different amounts of H2O2 and PtNPs were added to the detection cell to evaluate the feasibility of the device. As seen from Figure 2C, in the absence of H2O2 and PtNPs, the current change was nearly close to zero (curve a). Logically, a slight current change was observed with pure H2O2 because H2O2 was easy to decompose under ambient conditions (curve b). In contrast, when adding H2O2 and PtNPs together into the detection cell, the current change largely increased with a 9.5fold increase relative to that with H2O2 only. These results indicated that (i) the gas originating from H2O2 could be accelerated by PtNPs as the detection signal; (ii) the flexible pressure sensor was able to sensitively monitor the pressure change occurring by the decomposition of H2O2. Therefore, it was completely feasible to equip the as-prepared pressure sensor as a signal readout in the pressure-based POC testing. Under optimal conditions (Please see Figure S1 in the Supporting Information), the detectable ability and sensitivity of pressure-based POC testing were evaluated by assaying different concentrations of CEA standards. As seen from Figure 3A, the resistance change obtained from digital multimeter (DMM) was observed to increase gradually with the increasing target CEA in the sample. Meanwhile, a good linear relationship between the resistance change and the logarithm of target CEA was obtained within the dynamic range of 0.5−60 ng/mL (Figure 3A). The linear regression equation could be expressed as follow: ΔR (kΩ) = 32.78 × log CCEA + 30.31, (R2 = 0.992, n = 7), where the ΔR and CCEA were the resistance change of flexible pressure sensor and CEA concentration. The limit of detection (LOD) was calculated to be 0.167 ng/mL at a signal-to-noise ratio of 3. Furthermore, it
( )
δ S=
Figure 2. (A) TEM image of PtNPs (inset, photograph of PtNPs); (B) DLS of PtNPs; (C) current response of pressure sensor (a) without H2O2 and PtNPs, (b) with H2O2 (100 μL), and (c) with H2O2 (100 μL) and PtNPs (2 μL).
ΔI I0
δP
ΔI = I − I0
(1) (2)
where the S was the sensitivity, I was the current when pressure was applied, I0 was the current without applied pressure, and P was the applied pressure. As shown in Figure 1F, when the applied pressure increased from 0.08 to 50 kPa to the device, the current of the sensor increased correspondingly due to the enhancement of contact between the two paper electrodes. It was also important to note that the sensitivity was calculated as 0.077 kPa−1 in the low-pressure range and 0.012 kPa−1 in the high-pressure range. The explanation of the sensitivity change could be expressed that the increasing rate of the contact area between the two paper electrodes gradually slowed down with the increase of applied pressure, causing the saturation of C
DOI: 10.1021/acs.analchem.8b04635 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
based POC testing could be used for quantitative detection of the CEA concentrations of clinical serum samples. In summary, this contribution successfully demonstrated the development of advanced POC immunoassay based on paper electrode-based flexible pressure sensor with digital multimeter readout. This system was designed by integrating a typical immunoreaction as the pressure signal generator and a flexible paper-based pressure sensor as the signal readout. Compared with traditional immunoassays, there are several prominent advantages for the pressure-based immunoassay. Above all, a paper-based flexible pressure sensor is successfully fabricated with high response and sensitivity for tiny pressure change and used for developing a pressure-based POC testing as the novel signal readout. Second, in this detection system, the pressure change occurred by the decomposition of H2O2 was equipped as the detection signal, because an obvious change can be caused by a small amount of gas. Finally, target-triggered immunoreaction combining platinum nanozymes are introduced to amplify the signal to achieve sensitively detecting target. With such advantages, the pressure-based bioassay is enabled to implement facial, cheap, sensitive, and portable detection of important biomarkers and present a versatile assay protocol for point-of-care testing for remote area and daily life.
Figure 3. (A) Calibration curve of paper electrode-based flexible pressure sensors for POC immunoassay toward target CEA standards with different concentrations; (B) specificity of the POC testing toward CEA, CA 125, AFP, PSA, CA 15−3 (10 ng/mL used in these case).
was worth noting that the threshold value of CEA in normal human serum was around 3 ng/mL,30,31 which was within the detectable range. Thus, the proposed POC testing could sufficiently satisfy the requirement of the practical application in daily life. The reproducibility and precision of the pressure-based POC testing were studied by assaying three CEA standards (1.0, 10, and 30 ng/mL). As indicated by the result, the coefficients of variation (CVs) of the intra-assay were 8.2, 8.9, and 9.5% for above-mentioned three samples by using the same-batch nanostructures, respectively. Similarly, the CVs of interassay with various-batches nanostructures were 9.3, 10.1 and 11.4% for 1.0, 10, and 30 ng/mL, respectively. Hence, these results suggested that the reproducibility and precision of the proposed POC testing were acceptable. Furthermore, to evaluate the specificity of pressure-based POC testing for CEA, the detection system was studied by assaying other possibly interfering biomarkers in human serum, for example, cancer antigen 125 (CA 125), alpha-fetoprotein (AFP), prostate specific antigen (PSA), and cancer antigen 15−3 (CA 15−3). As shown in Figure 3B, the resistance changes of the pressure sensor were almost the same as the blank group in the absence of target CEA. In contrast, a strong resistance change was observed only in the presence of target CEA, regardless of being alone or mixed with others. In addition, the value of resistance change could remain about 95.3% of the initial response after being stored for 3 months. Thus, such high specificity and long-term stability were acceptable. The analytical accuracy and reliability were significant for the developed detection system with complex samples. To investigate this concern, eight human serum specimens containing CEA were collected from the Tumor Hospital of Fujian Province (Fuzhou, China), which were operated complying with the local ethical committee and relevant laws. CEA concentrations in these samples were monitored by the proposed pressure-based POC testing and calculated according to the above-mentioned calibration curve in Figure 3A. The CEA samples with high levels were diluted with PBS solution (pH 7.4) to below 60 ng/mL. For the reference, these samples were also measured by commercial CEA ELISA kit, respectively. The accuracy of the method was elucidated by the regression equation between the two methods, which was calculated with Y = 0.969X + 0.040 (R2 = 0.988, n = 8) (Please see Figure S2 in the Supporting Information). The slope and intercept close to “1” and “0”, respectively, revealed that no obvious divergences were observed between the two methods in analyzing the 8 human serum samples. Thus, the pressure-
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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.8b04635.
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Materials and reagents; preparation of platinum nanoparticles (PtNPs); preparation of PtNP-labeled detection antibody; preparation of the paper electrode-based flexible pressure sensor; preparation process of the flexible pressure sensor (Scheme S1); immunoreaction protocol and digital multimeter measurement; optimization of experimental conditions; effects of incubation time, PtNPs catalytic time, and H2O2 concentration on the response of the pressure-based POC testing; and comparison of the results for human serum specimens obtained by the pressure-based POC testing and human CEA ELISA kit (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail:
[email protected]. ORCID
Dianping Tang: 0000-0002-0134-3983 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Grants 21675029, 21874022, and 21475025).
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DOI: 10.1021/acs.analchem.8b04635 Anal. Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.analchem.8b04635 Anal. Chem. XXXX, XXX, XXX−XXX
