Triggerable H2O2–Cleavable Switch of Paper ... - ACS Publications

Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/ac

Triggerable H2O2−Cleavable Switch of Paper-Based Biochips Endows Precision of Chemometer/Ratiometric Electrochemical Quantification of Analyte in High-Efficiency Point-of-Care Testing Li Li,† Yan Zhang,† Shenguang Ge,‡ Lina Zhang,§ Kang Cui,*,† Peini Zhao,† Mei Yan,† and Jinghua Yu*,† †

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R. China Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan 250022, P. R. China § Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, P. R. China

Downloaded via KEAN UNIV on July 22, 2019 at 01:40:34 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: In this work, a triggerable H2O2-cleavable fluid switch mediated paper-based biochip, being amenable to multiplexing and quantitative analysis with the dual-response output of visual screening and ratiometric electrochemistry, was developed for sensitive detection of target on-site. By properly implanting hydrophobic Ag−H2O2 responsive material in specific zone to form a programmable fluid switch, the biochip could achieve different modes of blocking/connecting switching automatically. In order to improve the test performance, a ratiometric electrochemical signal readout was designed, which was enhanced by a secondary in situ growth method fabricating trepang-shaped Au modified paper working electrode. In virtue of hybridization chain reaction, classic competitive recognition interactions of aptamer and target, and ratiometric internally calibrated mechanisms, ultrasensitive detection of the target was realized. To acquire a more quantitative and straightforward naked eye visual screening, the hydrophobic Ag switch was triggered by stimulating instructions from H2O2, thus reconnecting the electrochemical and ratiometric units automatically and resulting in a “signal on” visual fluidic flow on the chemometer characterized by the accurate distance of color development as a detection motif. With MCF-7 and K562 cells as models, wider linear detection ranges from 150 to 1.0 × 107 and 220 to 7.0 × 106 cells mL−1 for MCF-7 and K562 cells, respectively, were achieved. Meanwhile, thanks to the paper fluid chemometer, an acceptable screening detection limit of 103 cells mL−1 was obtained in the quantitative colorimetric assays. The proposed paper-based biochips opened up new horizons for designing of integratable, easy-to-use, and precise point-of-care testing devices.

P

facilitated functionalization.10−14 As a matter of principle, to successfully develop and eventually achieve accurate quantification of analyte in a new POC testing, the key point is to employ simple and sensitive signal readout. By rationally designing the fluid transfer state,15−17 the paper-based devices readily join multiple functional units together, enable signal generated efficiently, and detected easily, especially through intuitionistic visual diagnostic readout. They have a great application potential for screening of specific targets, particularly in resource-limited regions where medical infrastructure is lacking. Despite some achievements, regulating the fluid delivery modality by unsophisticated transformations of steric configuration and channel geometry, these devices just offer a singlepoint visual output with a limited sensitivity, which are not able

oint-of-care (POC) testing device, featuring with miniaturization, portability, and accessibility, offers powerful tools for individualizing diseases diagnosis, in-home preventive care, as well as dealing with unexpected public health incidents.1−5 With the sustainable development and popularization of the POC analytical techniques, which drastically reduce diagnostic expense, save diagnostic time, and subvert dependency on trained professionals, a few kinds of hand-held microdevices have appeared on the consumer market, such as the pregnancy test strip and glucometer.6 Yet, this could not fulfill the increased demand of the present medical market. Given the realities, it is necessary to develop new protocols and strategies to explore a reliable, accurate, and easy-to-use diagnostic device for POC applications in clinical medicine. Paper-based biochips,7−9 as one of the representative POC device, have attracted substantial attention in the operation of POC diagnostics owning to the alluring nature of cellulose, such as abundant reserves, low cost, green biodegradation, and © XXXX American Chemical Society

Received: May 28, 2019 Accepted: July 9, 2019 Published: July 9, 2019 A

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Scheme 1. Three-Dimensional Structures of the Integrated Paper Chip (A); Enlarged Paper-Based Detecting Units (B); Size of Each Parts of the Biochip (C, D); (E) Photograph of the Bimodal Paper Chip Assembled with Circuit Boards

high efficient beforehand check of specimen. First, to achieve a sensitive electrochemical signal, secondary in situ growth method was first applied to prepare trepang-shaped Au modified paper working electrode (TSAu-PWE), thus effectively improving conductivity and notably enriching the effective area of electrode. By virture of hybridization chain reaction and classic competitive recognition interactions of aptamer and target, the target and cubic dendritic hollow (CDH) Pd−Pt nanoparticles (NPs) loading with ferrocene (Fc) were linked on the surface of TSAu-PWE. Meanwhile, the DNA strand labeled with methylene blue (MB) was released from the electrode surface. Then an increased ratio of current intensity of Fc/MB was achieved, and thus offering ultrasensitive detection of target. Second, a “H2O2-cleavable fluid switch of Ag” was tactfully grown on the all-in-one paper chips, realizing self-acting switching on the fluid channel and establishing correlativity with the target. Finally, visual reation would be triggered by the delivered H2O and thus, the quantification screening of target was achieved through the paper fluid chemometer. As-a-proofof-concept, MCF-7 and K562 cells were successfully sensitively quantified and visually detected. The proposed protocol offers a new strategy for quantification of various analytes, from environmental to clinical samples by simply altering the specific probes, in high-efficiency point-of-care testing, and greatly improves the practicability of paper-based biochips.

to nicely satisfy the required precision for screening targets with low abundance. Besides, most of the processing technologies on flow state regulation are always along with user-activated operatation, such as multistep folding process, which significantly limit the simplicity of paper-based devices and restrict the self-acting isolation and reconnection of multifunction area with programmed order on paper chips. Therefore, to render paperbased biochips measurements more quantitative and straightforward in POC diagnostics, subsequent work in the area should focus on the improvement of configuration technology. For example, integrating multiple inlets per detection region and programmatically controlling the paper capillary behavior may achieve improved test performance, for example, a higher sensitivity. Since the tunable-delay shunts was first reinvented and applicated for controlling fluidflow on paper device by Fu’s group,18 several paper fluidic tools, such as dissolvable sugar barrier, modification of optode materials,19 loading with organic solvents,20 introducing aptamer-cross-linked hydrogel,21 or user-manual pressing or pulling3,17,22,23 were reported to properly turn on/off the fluid delivery. There is no denying that these systems could achieve the simple and rapid POC testing based on the permeability alteration, but more versatile fluid switch designing with responsive materials are still needed for more general applicability. To provide a rapid and sensitive test information for trace amounts of targets, analytical techniques with low background and good anti-interference ability are required. Ratiometric techniques,24−29 applied to elctrochemistry with significantly increasing analytical efficiency,30 apparently, match this purpose. Given all that, a “H2O2-cleavable fluid switch of Ag” is first developed and built into the difunctional paper-based chips, including preliminary screening domain of the visual naked eye with enhanced efficiency and ultrasensitive quantitative domain of ratiometric electrochemistry with improved performance, for automatic actuating of different modules. To realize the desired difunctional paper-based biochip, indagation was primarily emphasized on (1) constructing electrochemical functional domain with self-calibration capability for improving signal readout sensitivity; (2) implanting a “H2O2-cleavable fluid switch of Ag” to direct the channel on or off automatically; (3) designing a visual paper fluid meter for

■

EXPERIMENTAL SECTION Synthesis of CDH Pd−Pt NPs. The CDH Pd−Pt NPs were prepared according to the previous reported article.31 To begin with, Pd cubic seeds were prepared using redox reaction between 1.43 mM of K2PdBr4 and 8.57 mM of ascorbic acid (AA) under the protection of 34.29 mM cetyltrimethylammonium chloride. The solution was stirred and heated to 100 °C for 5 h. Then, the obtained Pd cubic seeds were used as sacrificial templates to prepare CDH Pd−Pt NPs. Briefly, 50.0 mM of AA, 1 mM of Pd cubic seeds, and 2.0 mL of K2PtCl4 were uniformly distributed and then added into 13.0 mL of 20.0 mM cetyltrimethylammonium chloride with vigorous stirring at 100 °C for 5 h. The obtained CDH Pd−Pt NPs were repeatedly washed by deionized water. Fabrication of the Aptamer Labeled CDH Pd−Pt NPs. Briefly, 20 μL of activated aptamer (S1 or S2, the typical sequences were shown in Table S1, Supporting Information B

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Scheme 2. Construction Procedures (A−C) and Visual Response Mechanism (D) of the H2O2−Cleavable Fluid Switch Mediated TSAu Paper-Based Biochip

(SI)) and hairpin probe (HP) were added into the prepared CDH Pd−Pt NPs (18 mM). To ensure the tight connection between the aptamer and CDH Pd−Pt NPs, the above solution was maintained at 4 °C for 18 h. Then the obtained products were centrifuged for 15 min at 12 000 rpm and redispersed in 10 mM of Tris-HCl. Cell Culture. K562 and MCF-7 cells lines were separately cultured in a flask in Dulbecco’s modified Eagle’s medium (obtained from Gibco (Beijing, China)) including fetal bovine serum (10%) in a humidified atmosphere (containing 5% CO2 and 95% air) and incubated at 37 °C. Design of the Naked Eye Direct-Reading Paper Fluid Chemometer. The visual unit contains two layers of paper (Scheme 1), named chromogenic layer and transfer layer, respectively. On the chromogenic layer, 13 reservoirs (3 mm in diameter) were designed and equidistantly distributed on paper (Scheme 1D). Each reservoir was prestored with CuSO4 and they could be connected together once the fluid transferred from left to right side through the lower transfer layer (in length of 9 mm and 25 mm for the bilateral and the middle transfer layer, respectively) with fluid channel patterned on it. To reduce the loss of liquid during the transfer process, the fluid channel was designed to be semihydrophilic layer, in which the upper surface was hydrophilic and the lower surface was hydrophobic (pale yellow region). For achieving the multidirectional transfer of the solution simultaneously, a three-throw diverter (9 mm in diameter) was originally introduced on the separator layer. When the sample was added onto the surface of the paper through a hole above the diverter inlet, the solution would be

equally divided into three parts. Two of them arrived the bilateral working zones (8 mm in diameter) modifying with functional nanomaterials and the middle circular region was blank area (8 mm in diameter), which combined with the middle channel of transfer layer forming “fluid timer”. When the fluid timer was turned on (the five reservoirs all becoming blue), the transmission distances of the bilateral fluids were recorded. It should be noted that the reaction between water solvent and CuSO4 power (white) endowed the five reservoirs with blue color. Through the two chemometers for the MCF-7 and K562 cells, visual and high-throughput analysis of the cells can be obtained without any photo/electrosignal capturing devices. Besides, the used channel can be regenerated by soaking and further curing the chromogenic layer in oven at 60 °C for 2 h. Assembly Process of the Biochip and Cell Capture. Typically, the paper modified with Au seeds32 was added into a beaker containing 500 μL of HAuCl4 (1% wt) and 1 mL of NH2OH·HCl under shaking and reacted for 5 min. Then the second growth of Au was conducted using the above paper as the template. 40 μL of NH2OH·HCl and 26 μL of HAuCl4 (1 wt %) were blended under shaking to form uniform solution. Then the above solution was added onto the surface of the Au modified paper. After reacted for 10 min, the obtained TSAu-PWE was thoroughly rinsed. To immobilize the aptamer onto the working electrodes, 60 μL of 4.0 μM S1 and S2 solution were injected into the two working zones, respectively, and reacted at 37 °C for 2 h. After rinsing with phosphate-buffered saline (PBS, pH 7.4), the redundant active sites were covered with 6-mercaptohexanol (2 C

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry mM). After that, to conduct the hybridization reaction, 1 μM complement DNA of S1 and S2 (named C-S1 and C-S2) solution were added onto the above electrodes and incubated at 37 °C for 2 h, respectively (Scheme 2A). After drastically rinsed with PBS, cells capture were proceeded by adding 100 μL of MCF-7 and K562 cells with different concentrations and cultivated for 1 h (Scheme 2B). Following that, the electrodes were washed with distilled water. Preparation of the Biochip with Dual-Response Output of Electrochemistry and Naked Eye Visualization. 50 μL of aptamer labeled Pd−Pt NPs were added onto the above paper electrode and reacted at 37 °C for 1 h (Scheme 2C), followed by washing with deionized water for three times. Then the obtained electrodes were used to perform visual prescreening and electrochemical detection. The electrochemical experiment was conducted on a electrochemical workstation by the differential pulse voltammetry (DPV) method sweeping from −0.4 to 0.4 V. Prior to testing, the paper device was folded as shown in Scheme 1C. Six layers, including reference, separator, working, blocking, chromogenic, and transfer layer were stacked perpendicularly together from top to bottom. The upper three layers were conducted the electrochemical output. The three electrodes, named reference, counter, and working electrode are printed on the reference and working layers, respectively through screenprinting technology. With 12 mM of PBS (pH 7.4) dropped through the electrochemical injection ports on the reference layer, the three electrodes would be immersed in the electrolyte and connected together. After 3 min’ standing, the paper device was connected with the electrochemical workstation to collect the current signals. To obtain the intuitionistic detecting results, visualization measurements were proceeded subsequently. First, 360 μL 15% H2O2 solution was added into the paper through the colorimetric injection port (Scheme 1C) for three times, then the fluid in both side channel will orderly pass through the diverter, working zone with different amounts of mimetic peroxidase and cells modification, blocking layer (with Ag nanoparticles modification, the detailed preparation procedures were similar with previous reported work33), and arrive chromogenic zone eventually, while the fluid in the middle channel will go direct to the chromogenic zone to trigger the chromogenic reaction. Therefore, the middle channel was designed elaborately as the paper fluid timer, avoiding usage of additional timer. When these zones were all turned blue, it was the time to record the numerical values of both side channels.

Figure 1. SEM images of the SLAu-PWE (A,B) and TSAu-PWE (C,D); High magnifications of SLAu-PWE (Figure 1B inset) and TSAu-PWE (Figure 1D inset).

nanoscale structure of the Pd seeds with sizes around 16 nm. Then, the produced CDH Pd−Pt NPs were characterized by TEM and the obtained images were shown in Figure 2B,C. Obviously, well-defined cubic dendritic hollow-like Pd−Pt bimetallic NPs were observed. Furthermore, energy-dispersive spectroscopy mappings further confirmed spatial distribution of CDH Pd−Pt NPs (Figure 2D-F). It should be noted that the image in Figure 2F was rotated for 90 deg counterclockwise. To gain more detailed information about lattice fringe of the asprepared CDH Pd−Pt NPs, high-resolution TEM (HRTEM) tests were conducted. The d spacing of 2.00 Å (Figure 2G) was detected, which was in line with the previous reported results,34 indicating the successful preparation of the CDH Pd−Pt NPs. Estimation of Effective Area and Conductivity. To prove the obtained TSAu-PWE with unprecedented enlarged electroactive areas and enhanced conductivity, cyclic voltammetry and sheet resistance tests were carried out. As illustrated in Figure 3A−F, commendable linear correlations between the oxidation (Figure 3B, E)/reduction (Figure 3C, F) peaks current and the square of scan rate were obtainted. The effective areas of the SLAu-PWE (Figure 3A-C), TSAu-PWE (Figure 3DF), and usual seeded-growth method (directly adding dropwise growth medium onto the surface of the PWE without magnetic stirring) prepared Au-PWE35 were calculated with the following formulas:

■

RESULTS AND DISCUSSION Characterization of TSAu-PWE. The surface architecture transformation of the paper electrode during the growing process was traced by the scanning electron microscopy (SEM). As displayed in Figure 1A and B, the Au modified PWE through single in situ growth technique under shaking showed sheet-like structure (SLAu for short), which were uniformly distributed on the paper fibers. After secondary growth of the Au NPs, there were still many porous cavities involving in the cellulose fibers. Besides, a number of Au NPs, with average diameter around 40 nm, were dispersed on the surface of the SLAu-PWE (Figure 1C, D), forming enormously interconnected bridges for electrons transportation and affording a biocompatible, highly conductive substrate for linking of biomolecules. Characterization of CDH Pd−Pt NPs. Transmission electron microscopy (TEM) image in Figure 2A shows

i p = 2.69 × 105n3/2 AD01/2 ν1/2c0(25°C)

where ip was the oxidation (Ipa) or reduction peak current (Ipc), n was number of electron transferred (n = 1), A was the active area of the electrode (cm2), D0 was diffusion coefficient (D0 = 7.6 × 10−6 cm2·s−1), ν was scan rate (V·s−1), and c0 was bulk concentration of the redox probe. Hence, the effective area of the Au-PWE (SI Figure S1), SLAu-PWE, and TSAu-PWE were 0.5962 (8 mm in diameter), 0.7282 (8 mm in diameter), and 0.7943 cm−2 (8 mm in diameter), respectively. The electroactive areas were about 1.19, 1.45, and 1.58-fold compared with that of undecorated electrode (8 mm in diameter, 0.5026 cm−2). Therefore, the growth times increase and introduction of magnetic stirring make it possible to greatly enhance the active areas of the prepared working electrodes. D

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 2. TEM images of Pd nanocube seeds (A) and CDH Pd−Pt NPs (B,C); Elemental mapping for different elements in CDH Pd−Pt NPs (D-F); HRTEM of CDH Pd−Pt NPs (G).

Figure 3. Cyclic voltammetry curves of the (A) SLAu-PWE, (D) TSAu-PWE, (H) SLAu-PWE, TSAu-PWE, Au-PWE, and bare PWE in [Fe(CN)6]3−/4− (5.0 mM) solution containing 0.1 M of KCl with scan rates varied from 10 to 70 and 10 to100 mV s−1 for SLAu-PWE and TSAu-PWE, respectively; The corresponding linear relations of the SLAu-PWE (B,C) and TSAu-PWE (E,F) between ip and square root of scan rate; (G) Sheet resistances of the bare cellulose, screen-printed carbon, Au-PWE, SLAu-PWE, and TSAu-PWE; Pictures of the above electrodes (Figure 3G inset); (I) DPV responses of the bare cellulose, Au-PWE, SLAu-PWE, and TSAu-PWE in MB solution (5.0 mM).

0.179 (Ω·sq−1) of resistivity, which is 8.4, 16.76, and 168-fold lower than that of the SLAu-PWE, Au-PWE, and sreen-printed carbon, respectively, and such delectable conductivity of the TSAu-PWE has not been reported previously. In order to investigate the electrochemical properties of different electrodes (bare PWE, Au-PWE, SLAu-PWE, and

To compare the change of electrical conductivity under different conditions, the resistivity tests were proceeded and the obtained results were shown in Figure 3G. Compared with the bare cellullose (>20 MΩ·sq−1),10,13,36 the sheet resistances of sreen-printed carbon,3 Au-PWE, SLAu-PWE, and TSAu-PWE greatly decreased. Particularly, the TSAu-PWE exhibits about E

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 4. DPV responses of the prepared cyto-sensor before (a) and after different concentrations of MCF-7 cells (b, 103 and c, 5 × 106 cells mL−1) (A), or K562 cells (b, 103 and c, 106 cells mL−1) linked on the electrode surface (B); (C) The corresponding photographs of visual detecting results and (D) SEM images of Ag switches of the above cyto-sensors (D1−D3 are switches for MCF-7 cells and D4−D6 are switches for K562 cells). The reservior number represents serial number of reservoirs (from left to right: 1−13). Insets: Digital images of water drop on the Ag switches.

Figure 5. Current intensity-potential curves of the cyto-sensor tested under different concentrations of MCF-7 (A) (150, 103, 5 × 103, 5 × 104, 106, 5 × 106, and 107 cells mL−1) and K562 cells (D) (220, 103, 104, 105, 106, 3 × 106, and 7 × 106 cells mL−1); Calibration curves for quantification of MCF-7 (B) and K562 cells (E); The photographs of visual screening results for the MCF-7 (C) and K562 cells (F).

couple of boosted oxidation/reduction currents was observed for the TSAu-PWE (blue line) compared with that of bare PWE (black line), Au-PWE (red line), and SLAu-PWE (green line). A similar phenomenon was obtained from the DPV tests (Figure 3I). Thus, we daringly speculate that the prominent electro-

TSAu-PWE), cyclic voltammetry and differential pulse voltammetry (DPV) measurements were conducted in the solution of K3[Fe(CN)6] (5 mM, containing 0.1 M KCl) (Figure 3H) and 12 mM PBS (pH 7.4) solution containing 5.0 mM MB (Figure 3I), respectively. As shown in Figure 3H, a F

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

regression equation for the MCF-7 cells was expressed as IFc/IMB = 0.12lgcMCF‑7 − 0.26 and the corresponding correlation coefficient was 0.996 (n = 5). For the K562 cell, the linear equation is expressed as IFc/IMB = 0.13l gcK562 − 0.30 (R = 0.995, n = 5). Compared with the previous reported literature, our developed cyto-sensor revealed comparable linearity ranges and detection limits (SI Table S2).37,38 Additionally, the visual screening results under different dosages of cells were recorded via the paper fluid chemometer, and each concentration was measured three times (Scheme 1D and Figure 5C,F). The detecting ranges of 103−107 and 103−7.0 × 106 cells mL−1 were achieved for screening of MCF-7 and K562 (Figure 5C,F), respectively, testifying the feasibility of visual prescreen. Besides, it should be noted that each chemometer enables to work without powering by any electronic equipment. Furthermore, to make the detection result more intuitionistic, three common colors, including green, orange, and red, were further introduced into the chemometer. Once the cells with high concentration were added into the sensor, the fluid would reach the red zone, indicated that much more attention should be paid to this sample. Contrast to this, as small amounts of cells were introduced into the sensor, the fluid would stay in the green zone, suggesting the sample with safe concentration. Therefore, the chemometer proposed here might provide infinite utilization potentiality in cancer-related warning device in future. Detection of the MCF-7 and K562 cells in Real Samples. To further assess the applicability of the developed POC device in complicated biological samples, real clinical blood samples provided from Shandong Tumor Hospital were added into different concentrations of cells. Then, the samples were tested using our bimodality sensor, and the obtained results were compared with the ELISA method. As shown in SI Tables S3 and S4, our developed device revealed a good agreement with the reference method, indicating the promising potential of the sensor in future clinical determination. In addition, the selectivity of the developed dual-response output of biochips was investigated. Four kinds of cells, including normal human breast cells (MCF-10A), 293T, Hela, and MCF-7 cells were linked onto the surface of the working electrodes, and the collected ratios of IFc/IMB are shown in SI Figure S6. Almost no detectable current response of Fc were detected for the MCF-10A, 293T, and Hela cells, except the presence of target (103 cells mL−1 MCF-7 cells was selected as an example). And the visual chemometer also draw a similar conclusion. All these results indicated that our developed biochip possessed remarkable selectivity. Versatility Evaluation of the Bimodal Biochip. To appraise versatility and integratability of the developed bimodal biochip, other kinds of targets, including of metal ion (Pb2+ was selected as an example) and protein (thrombin was selected as an example) were tested using our developed biosensor, and the obtained results are shown in SI Figure S7. It should be noted that the biosensor establishment process was the same as the procedures mentioned above, and the aptamer sequences were similar to previous works.39,40 As shown in SI Figure S7A, without the targets added, the MB exhibits an obvious electrochemical signal; however, no Fc signal was detected. With the targets introduced into the electrode surface, an increased Fc and decreased MB response were detected. Interestingly, the naked eye visual stripes became shorter than the electrodes without targets modification (SI Figure S7B). Undoubtedly, these results prove that the proposed fluid switch

chemical performances of the TSAu-PWE can be assigned to the enhanced effective area and conductivity, which are crucial adjective factors for improving the monitoring sensitivity during the electrochemical measurements. Feasibility Evaluation of the Biochip. To demonstrate the practical applicability of the developed paper biochip with dual-response output properties, MCF-7 and K562 cells with three concentrations (MCF-7:0, 103, 5 × 106 cells·mL−1; K562:0, 103, 106 cells·mL−1) were selected as examples. As shown in Figure 4A,B, the MB exhibited the highest current response, and no peak of Fc was detected in the absence of the MCF-7 (Figure 4A, line a) or K562 cells (Figure 4B, line a). The current intensity of MB decreased and the Fc revealed enhancive current responses with the increasing concentration of cells (Figure 4A,B, lines b,c). Therefore, the above results clarify that the developed biochip can efficiently quantify multiple tumor cells via electrochemical method. In addition, visual detecting ability of the proposed chemometer was then studied. As shown in Figure 4C, with addition of the 15% H2O2 solution onto the surface of the electrodes decorating with increased dose of cells, the fluid transfer distance on the chemometer reduced gradually. It should be explained that a underlying reaction was existed in the paper, that was, Ag NPs could react with the H2O2 solution with the following formulas: 2Ag + H 2O2 → 2Ag + + 2OH−

With introduction of high-concentration cells, more CDH Pd− Pt NPs would be linked onto the working electrode surface (Figure 4A,B and Scheme 2C), resulting in increased H2O2 consumption on the TSAu-PWE (Scheme 2D). The slight residuary H2O2 decreased consumption of sealing reagent (Ag NPs) in the blocking layer. In other words, there were more unreacted sealing reagent remaining on the blocking layer, thus preventing fluid of the transfer layer from moving a longer distance in unit time (Scheme 2D). Therefore, H2O-driven chromogenic reaction of CuSO4 power exhibits decreased delivery distance (Scheme 2D and Figure 4C inset). Consequently, the fluid transfer distance is proportional to the number of the fixed mimetic peroxidase, which relies on the amount of anchored cells. Besides, to better comprehend the mensurable visual results, the reacted Ag switches were tracked and characterized by the SEM and contact angle testing technologies. As shown in Figure 4D (D1−D3), with the increment of MCF-7 concentrations, the quantity of unreacted Ag switch increased gradually and the corresponding contact angles enhanced from 0 to 93° (Insets in Figure 4D1−D3). The similar variation trends were obtained for the K562 cells (Figure 4D4−D6), which further testified that the Ag switch played significant role in building a strong relationship with the target amounts and the length of the naked eye visualization. Bimodality Electrochemical/Visual Chemometer Biochip. Under the optimized experimental conditions (SI Figure S2−S5), the dynamic ranges for detection of MCF-7 and K562 cells were examined. From cells concentration-current curves of the cyto-sensor recorded at −0.4−0.4 V (Figure 5A, D), welldefined linear relationships between the concentration of MCF7 (Figure 5B) (or K562 cells, Figure 5E) and logarithm of the IFc/IMB were observed in the range of 150−1.0 × 107 for MCF-7 (Figure 5A, B) and 220−7.0 × 106 cells mL−1 for K562 cells (Figure 5D, E). The detection limits for the MCF-7 and K562 cells were 117 and 140 cells mL−1, respectively. And the linear G

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

■

of Ag−H2O2 responsive in this work can be used to effectively reconfigure the fluidic path, which provides a universal protocol for bimodal detection of multiple targets and paves a way for multifunctional zone integration on the paper chip.

CONCLUSIONS In summary, a new paper-based biochip with precision of chemometer/ratiometric electrochemical dual-response output was first proposed and used for sensitive detection and preevaluation of target on-demand. A unique great breakthrough of this work was developed: the updated triggerable H2O2cleavable switch to direct the wettability of the paper channel automatically and designed paper fluid chemometer for realizing intelligent initiating visual screening. Besides, a high-performance TSAu-PWE was first prepared via a secondary in situ growth method and served as ratiometric electrochemical answering platform, showing noticeable enhancement of conductivity and effective area compared with that of previously reported Au paper electrode, which endowed the biochips with notably enhanced sensitivity and wide linearity range. Given the bimodule sensing mechanism proposed in this work, we anticipate that the more sensitive paper biochips may be developed for detection of other targets by optimizing configuration technology of chip, growing nanomaterials with core−shell or multihole structure on paper fibers, introducing neotype signal amplification strategy or identification/immobilization biomolecule, such as DNA machines and framework nucleic acid, etc. ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b02459. Materials and reagents, estimation of effective area of AuPWE, optimization of the experimental conditions, selectivity and versatility evaluation of biosensor, the sequences information, performances comparison of various analytical methods, and testing of real samples and capacity comparison (PDF)

■

REFERENCES

(1) Zhang, L.; Ding, B.; Chen, Q.; Feng, Q.; Lin, L.; Sun, J. TrAC, Trends Anal. Chem. 2017, 94, 106−116. (2) Choi, J. R.; Yong, K. W.; Tang, R.; Gong, Y.; Wen, T.; Li, F.; Pingguan-Murphy, B.; Bai, D.; Xu, F. TrAC, Trends Anal. Chem. 2017, 93, 37−50. (3) Zhang, Y.; Zhang, L.; Cui, K.; Ge, S.; Cheng, X.; Yan, M.; Yu, J.; Liu, H. Adv. Mater. 2018, 30, 1801588. (4) Dou, M.; Macias, N.; Shen, F.; Dien Bard, J.; Domínguez, D. C.; Li, X. EClinicalMedicine 2019, 8, 72−77. (5) Zhu, Z.; Guan, Z.; Jia, S.; Lei, Z.; Lin, S.; Zhang, H.; Ma, Y.; Tian, Z. Q.; Yang, C. J. Angew. Chem., Int. Ed. 2014, 53, 12503−12507. (6) Karim, O.; Rao, A.; Emberton, M.; Cochrane, D.; Partridge, M.; Edwards, P.; Walker, I.; Davidson, I. Prostate Cancer Prostatic Dis. 2007, 10, 270−273. (7) Yang, Y.; Noviana, E.; Nguyen, M. P.; Geiss, B. J.; Dandy, D. S.; Henry, C. S. Anal. Chem. 2017, 89, 71−91. (8) Tenda, K.; van Gerven, B.; Arts, R.; Hiruta, Y.; Merkx, M.; Citterio, D. Angew. Chem., Int. Ed. 2018, 57, 15369−15373. (9) Güder, F.; Ainla, A.; Redston, J.; Mosadegh, B.; Glavan, A.; Martin, T. J.; Whitesides, G. M. Angew. Chem., Int. Ed. 2016, 55, 5727−5732. (10) Ha, D.; Fang, Z.; Zhitenev, N. B. Adv. Electron. Mater. 2018, 4, 1700593. (11) Yang, W.; Cao, R.; Zhang, X.; Li, H.; Li, C. Adv. Mater. Technol. 2018, 3, 1800178. (12) Dong, L.; Xu, C.; Li, Y.; Pan, Z.; Liang, G.; Zhou, E.; Kang, F.; Yang, Q.-H. Adv. Mater. 2016, 28, 9313−9319. (13) Feng, J.-X.; Ye, S.-H.; Wang, A.-L.; Lu, X.-F.; Tong, Y.-X.; Li, G.R. Adv. Funct. Mater. 2014, 24, 7093−7101. (14) Kong, D.; Wang, H.; Lu, Z.; Cui, Y. J. Am. Chem. Soc. 2014, 136, 4897−4900. (15) Thom, N. K.; Lewis, G. G.; Yeung, K.; Phillips, S. T. RSC Adv. 2014, 4, 1334−1340. (16) Li, L.; Zhang, Y.; Zhang, L.; Ge, S.; Liu, H.; Ren, N.; Yan, M.; Yu, J. Anal. Chem. 2016, 88, 5369−5377. (17) Kong, Q.; Cui, K.; Zhang, L.; Wang, Y.; Sun, J.; Ge, S.; Zhang, Y.; Yu, J. Anal. Chem. 2018, 90, 11297−11304. (18) Toley, B. J.; McKenzie, B.; Liang, T.; Buser, J. R.; Yager, P.; Fu, E. Anal. Chem. 2013, 85, 11545−11552. (19) Gerold, C. T.; Bakker, E.; Henry, C. S. Anal. Chem. 2018, 90, 4894−4900. (20) Lewis, G. G.; DiTucci, M. J.; Phillips, S. T. Angew. Chem., Int. Ed. 2012, 51, 12707−12710. (21) Wei, X.; Tian, T.; Jia, S.; Zhu, Z.; Ma, Y.; Sun, J.; Lin, Z.; Yang, C. J. Anal. Chem. 2015, 87, 4275−4282. (22) Li, L.; Zhang, Y.; Zhang, L.; Ge, S.; Yan, M.; Yu, J. Sci. Bull. 2017, 62, 1114−1121. (23) Zhang, Y.; Li, L.; Zhang, L.; Ge, S.; Yan, M.; Yu, J. Nano Energy 2017, 31, 174−182. (24) Oomen, P. E.; Aref, M. A.; Kaya, I.; Phan, N. T. N.; Ewing, A. G. Anal. Chem. 2019, 91, 588−621. (25) Zhang, J.; Zhou, J.; Pan, R.; Jiang, D.; Burgess, J. D.; Chen, H. Y. ACS Sens 2018, 3, 242−250. (26) Ewing, A. G.; Bigelow, J. C.; Wightman, R. M. Science 1983, 221, 169−171. (27) Phan, N. T. N.; Li, X.; Ewing, A. G. Nat. Rev. Chem. 2017, 1, 0048. (28) Hao, Q.; Shan, X.; Lei, J.; Zang, Y.; Yang, Q.; Ju, H. Chem. Sci. 2016, 7, 774−780. (29) Ying, Z.-M.; Wu, Z.; Tu, B.; Tan, W.; Jiang, J.-H. J. Am. Chem. Soc. 2017, 139, 9779−9782. (30) Xiong, E.; Zhang, X.; Liu, Y.; Zhou, J.; Yu, P.; Li, X.; Chen, J. Anal. Chem. 2015, 87, 7291−7296. (31) Hong, J. W.; Kang, S. W.; Choi, B.-S.; Kim, D.; Lee, S. B.; Han, S. W. ACS Nano 2012, 6, 2410−2419. (32) Li, L.; Zhang, Y.; Liu, F.; Su, M.; Liang, L.; Ge, S.; Yu, J. Chem. Commun. 2015, 51, 14030−14033. (33) Li, W.; Li, L.; Ge, S.; Song, X.; Ge, L.; Yan, M.; Yu, J. Biosens. Bioelectron. 2014, 56, 167−173.

■

■

Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *Phone: +86-531-82767040; e-mail: [email protected]. . ORCID

Yan Zhang: 0000-0002-1936-4619 Shenguang Ge: 0000-0002-0537-6491 Kang Cui: 0000-0002-4947-4448 Mei Yan: 0000-0002-7509-4262 Jinghua Yu: 0000-0001-5043-0322 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the Taishan Scholars Program (ts201712048), the National Natural Science Foundation of China (21874055), the Major Program of Shandong Province Natural S cience Foundation (ZR2017ZC0124), and the project of “20 items of University” of Jinan (No. 333 2018GXRC001). H

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry (34) Yuan, Q.; Zhou, Z.; Zhuang, J.; Wang, X. Chem. Commun. 2010, 46, 1491−1493. (35) Ge, L.; Wang, S.; Yu, J.; Li, N.; Ge, S.; Yan, M. Adv. Funct. Mater. 2013, 23, 3115−3123. (36) Tobjork, D.; Osterbacka, R. Adv. Mater. 2011, 23, 1935−1961. (37) Wang, S.-S.; Zhao, X.-P.; Liu, F.-F.; Younis, M. R.; Xia, X.-H.; Wang, C. Anal. Chem. 2019, 91, 4413−4420. (38) Zheng, T.; Zhang, Q.; Feng, S.; Zhu, J. J.; Wang, Q.; Wang, H. J. Am. Chem. Soc. 2014, 136, 2288−2291. (39) Huang, Y.; Li, L.; Zhang, Y.; Zhang, L.; Ge, S.; Li, H.; Yu, J. ACS Appl. Mater. Interfaces 2017, 9, 32591−32598. (40) Yang, J.; Dou, B.; Yuan, R.; Xiang, Y. Anal. Chem. 2016, 88, 8218−8223.

I

DOI: 10.1021/acs.analchem.9b02459 Anal. Chem. XXXX, XXX, XXX−XXX