Inkjet-Printed Bioassays for Direct Reading with a ... - ACS Publications

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Technical Note

Inkjet-printed bioassays for direct reading with a multi-mode DVD/Blu-Ray optical drive Xiaochun Li, Maolin Shi, Caie Cui, and Hua-Zhong Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501870w • Publication Date (Web): 21 Aug 2014 Downloaded from http://pubs.acs.org on August 26, 2014

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Analytical Chemistry

Revised Manuscript for ac-2014-01732p (Technical Note) (Updated Aug. 13, 2014)

Inkjet-printed Bioassays for Direct Reading with a Multi-mode DVD/Blu-Ray Optical Drive Xiaochun Li,†,‡,* Maolin Shi, † Caie Cui† and Hua-Zhong Yu†,‡,* †

Key Laboratory of Advanced Transducers and Intelligent Control Systems (Ministry of Education and Shanxi

Province), Taiyuan University of Technology, Shanxi 030024, P. R. China ‡

Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

*

Corresponding authors: [email protected] (X.L.); [email protected] (H.Y.)

Abstract Compact disc-based bioassays have been developed as novel point-of-care (POC) tools for various applications in chemical analysis and biomedical diagnosis. For the fabrication of assay discs, the surface patterning and sample introduction have been restricted to manual delivery that is unfavorable for on-demand high throughput medical screening. Herein, we have adapted a conventional inkjet printer to prepare bioassays on regular DVD-Rs, and accomplished quantitative analysis with a multi-mode DVD/Blu-Ray optical drive in conjunction with free disc diagnostic software. The feasibility and accuracy of this method have been proved by the quantitative analysis of inkjet-printed biotin-streptavidin binding assays on DVD, which serves as a trial system for other complex, medically relevant sandwich-format or competitive immunoassays.

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Introduction Microarrays are important tools for biochemical research, including genomics, proteomics, and cell analysis, which can be also used for disease diagnosis and drug discovery. However, as expensive robotic spotters and laser fluorescence scanners are needed to fabricate and image the microarrays, their applications have been restricted to hospitals and centralized biomedical laboratories with advanced infrastructure.1-3 Therefore, the development of inexpensive technology for rapid molecular screening is vital to improve public health care, particularly for early-stage, point-of-care, population-based biomedical screening. Since Kido et al. proposed the concept of preparing microarrays on compact discs (CD) with inkjet printing techniques and reading them with ordinary computer drives at the beginning of this century,4 various fabrication and detection techniques have been explored worldwide by scientists and engineers to develop CD technology-based molecular analysis and medical diagnostic devices.5-8 However, little progress has been made on surface patterning and sample introduction beyond popularly adapted microfluidic protocols.6,8 Especially the printing of CDbased microarrays with conventional office inkjet printers has not yet been accomplished. We have recently developed a CD/DVD-based digitized molecular diagnostic technique by using an unmodified optical drive coupled with free disc-quality diagnostic software.8 Using this method, the quantitative analysis of different types of bioassays prepared on standard CD-Rs and DVD-Rs has been achieved, including DNA hybridization, antibody-antigen binding,9 ppb-level detection with pre-assembled DNAzymes,10 and a sandwich-format immunoassay for hCG quantitation.11 In these experiments, all the probe and target reagents are distributed into microfluidic microchannels on a PDMS (polydimethylsiloxane) chip by manual pipetting.8-11 Imadd et al. have created integrated “digital microfluidic compact discs” for microparticle and cell counting, one step further towards producing truly portable, low-cost and ubiquitous diagnostic devices.12 Maquieira and co-workers have created CD/DVD micro-immunoassays with a special bio-printing device from BioDot Inc.13 and achieved quantitative analysis by integrating an amplification/detection board with the DVD drive circuit to acquire analog signals directly from the photodiode.14 Recently, Ramachandraiah presented a new version of “Lab-onDVD” system and demonstrated its capability for rapid and low-cost HIV diagnostics by counting CD4+ cells isolated from whole blood;15 Kim et al. have described a micro total 2 ACS Paragon Plus Environment

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analysis system for molecular analysis of Salmonella, a major food-borne pathogen, by developing a centrifugal microfluidic device that integrates the three main steps of pathogen detection, DNA extraction, isothermal recombinase polymerase amplification (RPA), and detection onto a single disc.16 In this technical note, we report a convenient and reliable method to fabricate bioassays on optical disc surfaces by conventional inkjet printing with a commercial desktop printer without any modifications to either the hardware or software driver. In conjunction with the digital reading protocol using DVD diagnostic software programs, we were able to quantitatively analyze inkjet-printed “ink assays” to define resolution and reproducibility. As only commercialgrade, off-the-shelf electronic devices were used in this study, we targeted the development of rapid, inexpensive assays for on-site chemical analysis and point-of-care medical diagnostics. To prove the concept of this novel approach, DVD-based biotin-streptavidin bioassays were prepared and analyzed with a multi-mode DVD/Blu-ray optical drive.

Experimental Materials and Reagents The Sylgard 184 Silicone Elastomer kit was purchased from Dow Corning Corporation (Midland, MI). N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N’ethylcarbodiimide hydrochloride (EDC), N-morpholinoethane sulfonic acid (MES), bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Amine-PEG3-biotin (biotinyl-3,6,9trioxaundecanediamine) was purchased from Pierce Biotechnology Inc., nanogold®(1.4 nm diameter)-streptavidin conjugate was provided by Nanoprobes Inc.; deionized water (˃18.3 MΩ cm) was produced with a Barnstead Easy Pure UV/UF compact water system (Dubuque, IA). A silver enhancement reaction kit (LI Silver) was purchased from Nanoprobes Inc. (Yaphank, NY).

Printing and reading assay DVDs Video files were recorded on blank DVD-Rs (Sony Inc.) with Power2GO software (V6, Cyberlink). The DVD-Rs were activated in a UV/ozone cleaner (PSD-UV, Novascan Technologies Inc.) for 15 min. An Epson R270 printer with an external, continuous ink supply system (CISS) (Figure 1a) was chosen to prepare the DVD-based assays (vide infra). A free software program (PrintCD) was used to design the pattern of the assays and control the printing 3 ACS Paragon Plus Environment


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process. The assay DVDs were read with an external, multi-mode DVD/Blu-Ray optical drive (PLEXTOR, PX-LB950UE) (Figure 1b); a free disc-quality diagnostic program (PlexUTILITIES 1.3.3.1, http://www.plextoramericas.com/) was used to examine the error distributions (in both PIE/PIF modes) before and after the formation of bioassays.11,17 This program controls the optical drive to run at 16× speed, so that it typically takes 5 min to screen an entire DVD.

Formation of the biotin/streptavidin binding assays on DVD Figure 1(c) shows the general procedure of printing and reading biotin/streptavidin binding assays on a DVD-R. After rinsing with ethanol and deionized water, the polycarbonate (PC) surface of a regular DVD-R was activated for 15 min and incubated for 20 min in a UV/ozone cleaner, followed by treatment with 100 mM EDC and 25 mM NHS for 30 min. After activation the DVD-R was placed on the printing tray of an Epson R270 printer. All the nozzles were washed six times (under the control of the printer software) with deionized water. Then 500 µL of 1 µg/mL amine-PEG3-biotin in 0.1 M phosphate buffer (pH 7.0) was injected into the black inner ink cartridge, the yellow cartridge was filled with 1.2 μg/mL nanogold®-streptavidin in 20 mM phosphate buffer (pH 7.4, 150 mM NaCl, 4% BSA, and 2 mM NaN3), and the cyan cartridge was filled with 20 mM phosphate buffer. We designed eight bars (8 mm × 0.4 mm) with the Print CD software, and printed the amine-PEG3-biotin on the activated DVD-R five times. After the immobilization reaction, a PDMS chip was placed on the DVD-R surface which has 8 channels with the same pattern and dimension as the designed eight bars (8 mm × 0.4 mm). The PDMS chip position completely accorded with the amine-PEG2-biotin printed area, and the reaction zone was passivated by treatment with a 20 mM phosphate blocking buffer at pH 7.4 (containing 150 mM NaCl, 0.8% bovine serum albumin (BSA), 0.1% gelatin, 0.05% Tween 20 and 0.05% NaN3) for 15 min to reduce nonspecific adsorption. The nanogold®-streptavidin in the black chamber was diluted with the buffer in the cyan chamber; both were mixed on the surface in different proportions in the process of printing. After printing, the reaction zone was stained with the commercial Nanoprobes LI silver enhancement kit, for which the same PDMS channel plate as above was used to deliver the solutions. After incubating for 15 min, the PDMS chip was removed and the disc was cleaned by washing with 20 mM phosphate buffer.


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Results and Discussion Figure 1 shows the actual pictures of the office peripherals, an Epson R270 inkjet printer and a multi-mode external DVD/Blu-Ray optical drive for fabricating and reading the disc-based bioassays. The Epson R270 is a type of DOD (drop-on-demand) inkjet printer (Figure 1a) for which a droplet is ejected as a result of a pressure pulse formed by a piezoelectric actuator; therefore, the droplet in the nozzle chamber does not need to be heated. This is an important feature as we don’t have to worry about the thermal stability of the reagents being printed. The continuous ink supply system (CISS) (Figure 1a) has six external ink containers connected to the inner cartridges with a catheter; it can supply ink to the inner cartridges continuously. Another feature of the Epson R270 printer is the attached disc printing tray. By using the software “Epson PrintCD” provided by the manufacturer, it allows precise control of the design and printing process, including the selection of ink cartridges to withdraw solution, as well as the position, pattern, and times of repeated printing (to control the sample volume delivered to the surface). In our experiments, the reagent solution was used as the “ink” to be printed on the disc: we have confirmed that a minimum volume of 500 μL is needed for the printer to work, and that only 18.7 pL are needed for covering an area of 1 mm2. Although the fabrication of DVD-based bioassays with standard inkjet printers has not been reported, the inkjet printing technology has been adapted to deliver chemical and biological reagents for both sample preparation and device fabrication in modern analytical sciences.18-25 For an example, taking advantage of the drop-on-demand capabilities, inkjet printing has been explored for preparing combinatorial arrays of different matrix-assisted laser desorption / ionization (MALDI) matrices in multiple concentrations on a single chip.18-19 Morlock et al. have demonstrated a new approach using an elegant, simplified system assembled from ordinary inkjet printers and office scanners to perform separations on monolithic and nanostructured ultrathin-layer phases for high-performance thin-layer chromatography (TLC) experiments.20 Inkjet printing technology has also been extensively studied for the fabrication of paper-based sensors and devices for either colorimetric or spectroscopic reading.21-23 We have reported the fabrication of microsensors on gold CD-Rs by inkjet printing alkanethiolate SAMs followed by chemical etching;24 the same approach was used to fabricate microelectrode sets for immobilizing DNA strands and modulating their conformations.25 The first task is to examine the actual resolution of the ink spot assays printed on a photo5 ACS Paragon Plus Environment


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activated DVD-R. The printing resolution of the Epson R270 printer is specified as 5760 dpi × 1440 dpi, corresponding to a spot size of 4.4 μm (vertical) × 18.1 μm (lateral); though the minimum diameter (100 µm) which can be assigned by the software PrintCD is much bigger. An ink spot array with 53 spots of 100 µm diameter was printed on the DVD-R surface as our first test. As shown in Figure 2 (top inset), the printed spots are in fact much larger (296 ± 22 μm) than the designed dimension that is likely due to the diffusion of ink on the hydrophilic disc surface. When the DVD-R printed with the ink spot array was tested with the multi-mode DVD/Blu-Ray drive, a series of distinct peaks were observed by using the PlexUtilities error detection software (Figure 2a). Here the software was run in the PIF (parity inner failure) mode, and the x-axis shows the logical position (in MB) of each peak. In Figure 2(b), we have zoomed on 6 spots, and changed the x-axis of the plot from its logical position to radial distance (the distance from the DVD center) according to the following equation r=

x × (5.82 − 2.4 2 ) + 2.4 2 4489.25

(1)

where x is the logical position (MB), and r is the radial distance (mm). For a standard singlesided DVD-R, the data was recorded in the area between 24 mm and 58 mm radial distance. As shown in Fig. 2(b), not only the ink dots produced reproducible PIF counts, but also the size of the spots indicated by the peak width was uniform. The average size determined from the error plot (288 ± 35 μm) is consistent with the actual diameter of the spots measured on the optical image (top inset of Figure 2b). With the above successful tests on the resolution of the “ink dot assays”, we proceeded to the direct printing of biotin-streptavidin assays (Figure 1c). The first step was to print an aminePEG3-biotin solution on an activated DVD-R for probe immobilization. We followed the design pattern of the ink spot assay, i.e., we created a set of eight strips of identical size (8 mm×0.4 mm) and printed a 30 μM solution of amine-PEG3-biotin in 0.1 M phosphate buffer on the DVD-R. The repeated printing times (to control the reagent amount) were increased consecutively for every two strips from one to three, to five and to seven, respectively, from left to right. The target nanogold®-streptavidin (1.0 µg/mL) and the silver enhancement solutions were applied subsequently. The optical images of the silver-stained strips are shown in the top inset of Figure 3(a); with increasing numbers of printing times (amount of analyte solution), the binding strips became darker. 6 ACS Paragon Plus Environment

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For the purpose of comparison with traditional assay methods, we also determined the ODR (optical darkness ratio) value of each strip based on the optical image shown in Figure 3(a) (top inset). As an established colorimetric protocol,26 the ODR value is defined as ODR = ( I b − I s ) / I b

(2)

where Ib is the luminosity of the background and Is is that of the binding site.8-11,17,26 The ODR values obtained for the printed ink strips were plotted together with the PIF densities as function of the printing times (Figure 3(b)). It is important to note the very similar increasing trends for both PIF density and ODR value, both reaching their maximum values after five repeated printings. Five times of repeated printings produced satisfactory (saturated) signal, indicating that this reagent amount is adequate for probe (amine-PEG3-biotin) immobilization. After addressing the key issues of printing resolution and quantity of reagent delivery, we fabricated the bioassays with repeated binding strips on a DVD-R to test reproducibility. Figure 4 shows the results for eight identical biotin-streptavidin binding strips on DVD-R with the same concentrations of nanogold®-streptavidin conjugates (1.0 µg/mL). In addition to the optical image of the assay (top inset, Figure 4a), we have shown the error reading plot (Figure 4a) and the PIF density (Figure 4b) with respect to the physical position of the binding strips. The eight replicated biotin-streptavidin binding strips show distinct and reproducible signals, particularly the PIF densities (372±17 counts/mm2) are within a relative uncertainty of less than 5%. This result confirms the high experimental precision of creating binding assays by direct inkjet printing with an office printer. With the fully adapted ink-jet printing protocol, strips of different concentrations of nanogold®-streptavidin conjugates were prepared by delivering them to the respective binding sites from different “ink” cartridges. The strip labeled as “0.0” was printed by the “ink” in the cyan box (buffer solution only) (top inset, Figure 5(a), as an example). The distribution of PIF counts on the DVD corresponding to each sample is shown in Figure 5(a). In Figure 5(b), we have shown both the PIF densities and ODR values of the printed binding strips as function of the target (nanogold®-streptavidin conjugate) concentrations; the consistency trends between the PIF and ODR values confirm the validity of using digital error reading methods for the assay quantitation. Initially, both PIF and ODR were approximately proportional to the target concentration, and then became saturated when the concentration reached 1.0 µg/mL. Without



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further optimization, the detection limit of 0.2 µg/mL and response range of 0.2 – 1.0 µg/mL are comparable with the binding assays prepared by the standard microfluidic approach.8-9 While more complex, sandwich-format or competitive immunoassays shall be created and tested to further validate the technology, we have demonstrated the simple and rapid fabrication and test of DVD-based bioassays by using an office inkjet printer and a multi-mode DVD/Blu-Ray optical drive without any hardware and software modification. Due to limitations of the PrintCD software and the solution diffusion on the disc surface, the printed minimum dimension is about 300 µm; future studies should be carried out to decrease the printing size for improving the lateral resolution, so that higher density assay arrays can be produced on a single disc. The other limitation is the number of samples that can be analyzed (currently restricted by the number of ink cartridges); for the same reason the assay blocking and silver staining steps were accomplished with the traditional microfluidic approach (with the help of a PDMS channel plate). It is feasible to build an automated, continuous ink supply system with multiple sets of cartridges to tackle this limitation. Different from the trial biotin-streptavidin assays for which the binding kinetics is rather fast, other slow immunoassay reactions may also impose technical challenges for the inkjet printing assay technology.

Conclusion In this paper, a commercial ink-jet printer without any modification is adapted to print bioassays on DVD. An unmodified, conventional DVD/Blu-Ray optical drive was used for assay reading, and free disc-quality analysis software for data processing. For the inkjet-printed bioassays, the resolution is below 300 µm, and the optimum number of repeated printings was determined to be five times. By using the black cartridge of the continuous ink supply system, precise quantities of the probe solution were automatically delivered onto the DVD-designated areas, and different concentrations of target solutions were automatically introduced from the “color cartridges” controlled by the PrintCD software. The feasibility and accuracy of this method has been ultimately proved by the quantitative analysis of biotin-streptavidin binding, which is one of the most studied biological recognition reactions.


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Acknowledgement We gratefully acknowledge the financial support from the Natural Sciences and Engineering Research Council (NSERC) of Canada; the research was also jointly supported by the Natural Science Foundation of China (Grant No. 61174010), Shanxi provincial government (“100-talents program”), Shanxi international cooperation project (Grant No. 2012081043), and Shanxi Scholarship Council (Grant No.2013-038).We thank Dr. Eberhard Kiehlmann for editing the manuscript, Dengxue Qu and Xuejuan Meng for preliminary experiments.

Supporting Information Additional experimental details of printing and reading disc-based assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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(a)

(b)

Continues ink supply system (CISS)

Disc printing tray

(c)

DVD-R

(1) Probe immobilization

DVD assay

(2) Target binding

Silver particles

− COOH − COOH

UV/ozone 15 min − COOH

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EDC /NHS Amine-PEG3-biotin

Blocking

Ag+(aq)

Nanogold®-Streptavidin

Ag(s)

Figure 1. (a) Epson R270 inkjet printer with a continuous ink supply system (CISS) used to print the bioassay DVD. (b) Multi-mode DVD drive used to read the bioassay DVD. (c) Experimental procedure of printing biotin/streptavidin binding assays on a DVD-R with an office inkjet printer. The blocking and signal enhancement steps were carried out with the help of a PDMS channel plate (see main text for details).



(a)

PIF (counts)

5 4 3 2 1 0 0

1000

2000

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(b)

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3 2 1 0 25

26

27

28

Radial distance (mm)

Figure 2. (a) PIF distribution of the printed arrays of 53 ink-spots with the diameter of 100 µm. The top inset is an optical image of the ink spots. (b) PIF distribution of the first six printed ink-spots. The top inset is the enlarged view of the first six ink spots.


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(a)

1mm

50

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(b) 160

0.3 120 0.2 80

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2 PIF density (counts/mm )

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0.1

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0

0.0 0

1

2

3

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5

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Figure 3. (a) PIF distribution of printed biotin-streptavidin binding strips with different

printing times. The top inset is an optical image of the binding strips; (b) PIF densities and ODR values of the spot array as a function of printing times. The dash line is to guide the eyes only.



(a) 1mm

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400

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Figure 4. (a) PIF distribution of the 8 printed biotin/streptavidin binding strips with the

same nanogold®-streptavidin concentration (1.0 µg/mL). The top inset is an optical image of the printed binding strips. (b) PIF densities of the eight replicate strips with respect to their physical locations on the disc.


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(a)

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0.1

0.0 0.0

0.3

0.6

0.9

1.2

Concentration (μg/mL) Figure 5. (a) Quantitation of printed biotin/streptavidin binding assays; distribution of

PIF on a DVD-R with six printed biotin/streptavidin binding strips. The top inset is an optical image of the binding strips. (b) PIF density and ODR values as function of the target concentration; the dashed line is to direct the eyes only.



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