Electronic Supplementary Information. Electrochemical Immunoassay on 3D Microfluidic Paper-based Device

Transcription

1 Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2011 Electronic Supplementary Information Electrochemical Immunoassay on 3D Microfluidic Paper-based Device Dejin Zang, Lei Ge, Mei Yan, Xianrang Song and Jinghua Yu* Pretreatments of MWCNTs and chitosan The MWCNTs were firstly treated with 3:1 H 2 SO 4 /HNO 3 under sonication for 4 h to shorten the MWCNTs, remove metallic and carbonaceous impurities, and generate carboxylate groups on the MWCNTs surfaces (Lai et al., 2009). The resulting MWCNTs were separated and washed repeatedly with distilled water by centrifugation until the ph 7 was reached. A 0.25 mg ml -1 chitosan stock solution was prepared by dissolving chitosan flakes in hot (80~90 C) aqueous solution with 0.05 M HCl. After the solution was cooled to room temperature, the ph was adjusted to 3.5~5.0 with NaOH solution. The chitosan solutions were filtered using a 0.45 µm Millex-HA syringe filter unit (Millipore) and stored in a refrigerator (4 C) when not in use.

2 Fabrication of the 3D microfluidic paper-based electrochemical device Scheme S1 The schematic representation of the fabrication of Paper-A and Paper-B in bulk. (1) Wax penetrated Sheet-A and Sheet-B; (2) Sheet-A and Sheet-B with screen-printed electrodes. Wax was used as the paper hydrophobization and insulation agent to pattern Sheet-A and Sheet-B. Briefly, as shown in Scheme S1, the wax-patterns were printed on paper sheets in bulk by wax-screen-printing method 1. The wax-printed paper sheets were then placed on a digital hot plate set at 130 for 150 s, thus the wax melted and penetrated through the thickness of the paper to form the hydrophobic patterns. The patterned paper sheets were ready for screen-printing electrodes after removing the paper sheet from the hot plate and allowing it to cool to room temperature (<10 s). Detail procedures of screen-printing electrodes were described below. For Sheet-A, as shown in Scheme S1(2), eight working electrodes containing the wires and contact pads were screen-printed in the defined area on Sheet-A using carbon ink. The working electrodes were all

3 aligned to the paper working zones. For Sheet-B, a counter electrode and a reference electrode were screen-printed in the defined auxiliary zone on Sheet-B using carbon ink and Ag/AgCl ink respectively. After that, Sheet-A and Sheet-B were cut to Paper-A and Paper-B with the same size (Scheme S1). Fabrication of 3D microfluidic paper-based electrochemical immunodevice Scheme S2 Schematic representation of the fabrication procedure for this 3D-μPEID. (1) wax-patterned paper working zone layer after capture antibody/chitosan/mwcnts modification, blocking and washing; (2) after capturing and washing; (3) after incubation with signal antibodies and washing; A) BSA/capture antibody/chitosan/mwcnts modified paper working zone on the back of B) screen-printed carbon working electrode. The 3D microfluidic paper-based electrochemical immunodevice (3D-μPEID) was constructed by immobilizing the corresponding immunoarrays into the paper working zones on the back of screen-printed working electrodes through MWCNTs modification, chitosan coating and glutaraldehyde cross-linking as shown in Scheme S2. First, 4 µl carboxylated MWCNTs were applied to each paper working zone and dried at room temperature. Then, 3 μl of 0.25 mg ml -1

4 Electronic Supplementary Material (ESI) for Chemical Communications chitosan was coated into each MWCNTs modified paper working zone and dried in the air. After activating with 2.5% glutaraldehyde (4 μl, PBS) for 2 h and washed with PBS, 2 μl of 20 μg ml-1 different capture antibodies were dropped into the corresponding paper working zone, respectively, and incubated at room temperature for 20 min, subsequently, physically absorbed excess antibodies were rinsed (Detail washing procedures were described below) with PBS and washing buffer, and a drop of 20 μl blocking buffer was applied into each paper working zone and incubated for 20 min at room temperature to block possible remaining active sites against nonspecific adsorption. After washing with PBS, the resulting 3D-μPEID was obtained and stored at 4 in a dry environment prior to use. Detail washing procedure for this 3D-μPEID Fig. S1 Pictures of the washing procedures for this 3D-μPEID. A) Adding PBS or washing buffer to the back of the paper auxiliary zone while bending the paper to a inverted-u type; B) Changing the bend axis, indicated by the red arrow, to repeat the washing procedures; C, D) Contacting the two paper working zones with a piece of U-type blotting paper.

5 The detailed washing procedures was as follows: Due to the front and back surfaces of the wax-patterned paper electrochemical cells are open to atmosphere, thus the paper working zones on Paper-A can be washed by adding PBS or washing buffer to the front of the centre of Paper-A while bending the paper layer to a inverted-u type as shown in Fig. S1A. Then a piece of U-type blotting paper was contacted with the eight paper working zones simultaneously (Fig. S1C, D). The washing buffer goes through the paper and migrates along the paper channels by the capillary and gravity action to wash the paper channels and paper working zones and carries the unbound reagents with it into the blotting paper. This effective washing procedure was used in this work consistently and acquiescently. The washing process was important for preventing the nonspecific binding and achieving the best possible signal-to-background ratio. Another purpose for this washing procedure was to stop the incubation reaction at exactly same time. This washing procedure was repeated twice by changing the bend axis, indicated by the arrow in Fig. S1A, B, to make sure the washing was performed completely. For Paper-B, the washing procedure can be performed according to method proposed by Cheng et al. 2.The printed electrodes will firmly attach to the paper surface due to the penetration of binding reagents in the inks into the paper matrix. And they will not break or peel off from the device upon washing and folding 3. Electrochemical assay procedure of this 3D-μPEID The electrochemical assay procedures of this 3D-μPEID were shown in Scheme S2 and S3. To carry out the immunoreaction and electrochemical measurement, 2 μl sample solution containing different concentrations of AFP, CA-125, CA-199 and CEA in 10.0 mm ph 7.4 PBS was added to corresponding paper working zone and allowed for incubation at room temperature,

6 followed by washing with PBS or washing buffer according to the procedure mentioned above. Then HRP-labeled AFP, CA-125, CA-199 and CEA signal antibody (2 μl, 10 μg ml -1 ) was added to corresponding paper working zone, and allowed for incubation at room temperature. Scheme S3. Schematic representation of the assay procedure for 3D-μPEID. a) the facile device-holder; b) Paper-A was placed face down onto Board-A; c) Paper-B was placed face up onto Paper-A; d) After clamping the device-holder, electrochemical reaction in each paper working zone were triggered sequentially with the aids of a section-switch; e) circuit schematic of the section-switch. After washing with PBS or washing buffer again, for electrochemical assay, the 3D-μPEID was integrated with a newly designed facile device-holder (Scheme S3), which was used to fix and connect the 3D-μPEID to the electrochemical detector. This folder-type device-holder was comprised of two simple circuit boards name Board-A and Board-B, with conductive pads on them. Firstly, Paper-A was placed face down onto Board-A. Then the Paper-B was placed face-up onto Paper-A. After that, the device-holder was clamped to make the 3D-μPEID stacked closely. Ultimately, 20 μl 10.0 mm ph 7.4 PBS buffer were adding into the paper electrochemical cell on through the hole in Board-B. With the aid of a section-switch, eight working electrodes were

7 sequentially placed into the circuit to trigger the electrochemical reaction. The differential pulse voltammetric (DPV) measurements were performed. The electrochemical signals were measured using a portable electrochemical workstation (PalmSens Electrochemical Portable Apparatus) at room temperature. Optimization of pipetting volume Fig. S2 The influence of pipetting volume on the uniformity and sufficiency of protein immobilization (4 mm diameter paper zones); Reducing the pipetting volume of solutions into paper working zones would decrease the analytical cost remarkably by saving the reagents. Briefly, FITC-labeled CEA capture antibodies were used as a model to investigate the optimum pipetting volume of solutions into each paper working zone. The investigation procedures included (i) immobilization of the FITC-labeled CEA capture antibodies with different volumes on the chitosan/mwcnts modified paper working zone through glutaraldehyde cross-linking into paper working zones; (ii) then the paper working zones were washed twice following the procedure mentioned above; (iii) the fluorescence images of the paper channels and paper working zones were investigated on an inverse fluorescence microscope (ChangFang CFM-500E, China). Fig. S2 showed that when the pipetting volume was 2.0 μl, the uniformity and sufficiency of antibodies in the paper working zone was obtained. Thus, considering the analytical cost, 2.0 μl was selected as the optimal pipetting volume of all reagents. In addition, the small pipetting volume could further avoid the outflow of the solutions from paper

8 working zones to the paper channels, this could further reduce the cross-talk between adjacent electrodes and the nonspecific adsorption in the paper channels to increase the signal-to-background ratio and the sensitivity of this 3D-μPEID. Optimization of incubation time The incubation time is an important parameter affecting the analytical performance and time efficiency of POCT. At room temperature, the DPV responses increased with the increasing incubation time used in sandwich-type immunoassay and then leveled off, which indicated a saturated binding in the immunoreaction. The optimal total incubation time of AFP, CA-125, CA-199, and CEA immunocomplexes was 240 s, 250 s, 240 s and 230 s, respectively. A successful development of the multiplex immunoassay required that the common incubation time must be suitable for all analytes. Thus, a total incubation time of 240 s was selected in the further study. The incubation process on this 3D-μPEID needed shorter time compared with 1-3 h at 37 C for the traditional electrochemical immunoassay. This is partly due to the high surface-to-volume ratio, incompact porous structure and the small volume of the chitosan/mwcnts modified paper working zone. The immunoreagents diffused only short distances to react with each other. Furthermore, as the solutions dry in paper working zones, the concentration of each reagent increases; this concentration maybe further enhance the binding kinetics of antibody antigen 2. Finally, the short incubation times could be favorable to high sample throughput and rapid POCT.

9 Evaluation of cross-talk and cross-reactivity Fig. S3 Electrochemical responses for different antigens on different electrodes. A) AFP capture antibodies/chitosan modified working electrodes; B) CA-125 capture antibodies/chitosan modified working electrodes; C) CA-199 capture antibodies/chitosan modified working electrodes; D) CEA capture antibodies/chitosan modified working electrodes. Elimination of the cross-talk between adjacent paper working zones is essential in the multiplexed electrochemical POCT. According to the design of the electrochemical cells of Paper-A (Scheme 1), the diffusion of antigens and signal antibodies from one paper working zone to another should go through two long paper channels, and the washing direction could effectively exclude this diffusion. Hence, the possible cross-talk could be avoided. To further confirm the resistance to cross-talk, the cross-reactivity between antigens and non-cognate antibodies was also investigated. For the evaluation of cross-reactivity, this 3D-μPEID was incubated with the four different capture antibodies for 1.0 ng ml -1 AFP, 1.0 U ml -1 CA-125, 1.0 U ml -1 CA-199 and 1.0 ng ml -1 CEA, separately, following incubation in the mixture of four trace tag solutions. As expected in Fig. S3, only the paper working zone prepared with corresponding capture antibody showed an obvious DPV response. So the cross-reactivity between antibodies and nonspecific binding at the paper working zones could be eliminated.

10 Analytical performance Fig. S4 Calibration curves for AFP, CA125, CA199 and CEA. Under optimal conditions, the analytical performance of this method was verified by applying 2.0 μl of samples of human AFP, CA-125, CA-199, and CEA standard solutions at various concentrations in 10.0 mm ph 7.4 PBS. Both calibration plots showed good linear relationships between the peak currents and the logarithm values of the analyte concentrations in the range from ng ml -1 for AFP, 0.02 to 85.0 U ml -1 for CA-125, 0.05 to U ml -1 for CA-199, and 0.05 to ng ml -1 for CEA that covered most of the levels in human plasmas and serums was also observed (Fig. S4). The linear regression equations were I = 2.648lgc AFP (ng ml -1 ) (R = ), I = 2.539lgc CA-125 (U ml -1 ) (R = ), I = 2.493lgc CA-199 (U ml -1 ) (R = ), I = 2.641lgc CEA (ng ml -1 ) (R = ). The limits of detection for AFP, CA-125, CA-199, and CEA in standard solutions were 0.01 ng ml -1, 6.0 mu ml -1, 8.0 mu ml -1, and 5.0 pg ml -1 at a signal-to-noise ratio of 3σ (where σ is the standard deviation of a blank solution, n = 11), respectively. In addition, the limit of detection

11 at a signal-to-noise ratio of 3σ were 14.0 pg ml -1 for AFP, 11.0 mu ml -1 for CA-125, 13.0 mu ml -1 for CA-199, and 9.0 pg ml -1 for CEA in human serum samples. Thus, on the basis of this standard curve, this 3D-μPEID should be useful for the determination of the four tumor markers in real serum samples, due to the cutoff values of the four tumor markers in clinical diagnosis are 25 U ml -1, 35 U ml -1, 35 U ml -1 and 5 ng ml -1, respectively 4. Table S1. Assay results of real human serum by the proposed and reference method AFP concentration (ng ml -1 ) CA-125 concentration (U ml -1 ) Samples Proposed Reference Relative error (%) Proposed Reference Relative error (%) CA-199 concentration (U ml -1 ) CEA concentration (ng ml -1 ) Samples Proposed Reference Relative error (%) Proposed Reference Relative error (%) * Average of eleven measurements. The analytical reliability and application potential of this 3D-μPEID was evaluated by assaying clinical serum samples using the proposed method as well as the reference values obtained by commercially used full-automatic electrochemiluminescence immunology analyzer (Roche Cobas E601) in Cancer Research Center of Shandong Tumor Hospital. When the levels of tumor markers were over the calibration ranges, serum samples were appropriately diluted with 10.0 mm ph 7.4 PBS prior to assay. The results were shown in Table S1, the agreement between the two methods was acceptable. This method showed satisfied recoveries (n = 11) of 96 to 103%,

12 98 to 107%, 98 to 110% and 97 to 105% for AFP, CA-125, CA-199 and CEA, respectively. Hence, the developed 3D-μPEID provided a possible application for the simultaneous detection of AFP, CA-125, CA-199, and CEA in clinical diagnostics. Regeneration and Reproducibility of this 3D-μPEID This 3D-μPEID could be regenerated, due to the stably covalent immobilization of antibodies in paper working zones, for reuse by a simple and effective procedure with the elution reagents, which is very important for the further development of 3D-μPEID in low-cost application. Furthermore, the regeneration must avoid being harmful to the activity of the immobilized antibodies and damaging the bonds between the antibodies and paper working zone when the dissociation of the immunocomplexes occurs 5. Different elution reagents were tested using 5.0 ng ml -1 CEA as a model for regeneration purposes, such as salt solution of organic solvent, high concentration, buffer with low ph value, and diluted alkali solution of different concentrations and ph, were tested. The regeneration efficiencies (REs), proposed by Yakovleva et al. 6, were calculated according to the following equation. RT B RE = [ 1 ] 100% T Where, RT represents the DPV response obtained after the regeneration cycle, B is the DPV response for blank, and T is the DPV response before applying any regeneration step. 0.1 M glycine-hcl (ph 2.1) showed the best regeneration efficiency at more than 96% for these four cancer markers, which was chosen as the elution reagent for the regeneration of this 3D-μPEID. With the regeneration procedure, this 3D-μPEID could be used for tent cycles with an acceptable reproducibility.

13 The relative standard deviation (RSD), using CEA as a model, for ten parallel measurements in the same one paper working zone (intraassay) incubated with the incubation solution containing 5.0 ng ml -1 CEA was 4.74%, indicating a good precision. The detections of 5.0 ng ml -1 CEA on ten different Paper-As fabricated independently (interassay) showed a RSD of 3.23%, giving an acceptable fabrication reproducibility of this 3D-μPEID. When this 3D-μPEID was stored dry at 4 C (sealed) and measured at intervals of 3 days, No obvious change was observed after storing for 4 weeks, indicating that this 3D-μPEID was stable for storage or long-distance transport in remote regions and developing countries. References 1 (a) W. Dungchai, O. Chailapakul and C. S. Henry, Analyst, 2011, 136, 77-82; (b) S. Wang, L. Ge, X. Song, J. Yu, S. Ge, J. Huang and F. Zeng, Biosens. Bioelectron., 2011, DOI: /j.bios C.-M. Cheng, A. W. Martinez, J. Gong, C. R. Mace, S. T. Phillips, E. Carrilho, K. A. Mirica and G. M. Whitesides, Angew. Chem. Int. Ed., 2010, 49, Z. Nie, F. Deiss, X. Liu, O. Akbulut and G. M. Whitesides, Lab Chip, 2010, 10, T. X. Li Modern Clinical Immunoassay, Beijing, Military Medical Science Press, 1st edn, J. Yakovleva, R. Davidsson, M. Bengtsson, T. Laurell and J. Emnéus, Biosens. Bioelectron., 2003, 19, J. Yakovleva, R. Davidsson, A. Lobanova, M. Bengtsson, S. Eremin, T. Laurell and J. Emnéus, Anal. Chem., 2002, 74,