From Disc Activation to Multiplex Detection - ACS Publications

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Blu-ray Technology-Based Quantitative Assays for Cardiac Markers: from Disc Activation to Multiplex Detection Samuel Weng, Xiaochun Li, Michelle Niu, Bixia Ge, and Hua-Zhong Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01604 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 13, 2016

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Analytical Chemistry

Manuscript ID: ac-2016-01604g (Revised June 2, 2016)

Blu-ray Technology-Based Quantitative Assays for Cardiac Markers: from Disc Activation to Multiplex Detection Samuel Weng,† Xiaochun Li,*,‡ Michelle Niu,§ Bixia Ge,§ and Hua-Zhong Yu*,†,‡ †

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

‡

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

and Shanxi Province), College of Physics and Optoelectronics, Taiyuan University of Technology, Shanxi 030024, P. R. China §

eSenso Biotech Inc., 308-2999 Underhill Ave., Burnaby, British Columbia V5A 3C2, Canada

Corresponding authors. * E-mail: [email protected] (X.L.) [email protected] (H.Y.)

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Abstract Acute myocardial infarction (AMI) is the leading cause of mortality and morbidity globally. To reduce the number of mortalities, reliable and rapid point-of-care (POC) diagnostics of AMI are extremely critical. We herein present a Blu-ray technology-based assay platform for multiplex cardiac biomarker detection; not only off-the-shelf Blu-ray discs (BDs) were adapted as substrates to prepare standard immunoassays and DNA aptamer/antibody hybrid assays for the three key cardiac marker proteins (myoglobin, troponin I and C-creative protein), but also an unmodified optical drive was directly employed to read the assay results digitally. In particular, we have shown that all three cardiac markers can be quantitated in their respective physiological ranges of interest, and the detection limits achieved are comparable with conventional enzyme-linked immunosorbent assay (ELISA) kits. The Blu-ray assay platform was further validated by measuring real-world samples and establishing a linear correlation with the simultaneously obtained ELISA data. Without the need to modify either the hardware (Blu-ray discs and optical drives) or the software driver, this assay-on-a-BD technique promises to be a low-cost user-friendly quantitative tool for on-site chemical analysis and POC medical diagnosis.


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1. Introduction In the past two decades, both scientists and engineers have been exploiting consumer electronics (e.g., office scanner, disc player, and smartphone) as alternative cost-effective and user-friendly on-site analytical and point-of-care (POC) diagnostic tools to compliment centralized laboratory facilities and intrumentation.1,2 Particularly, a wide range of analytical devices have been developed based on optical disc and recording technology for various applications in areas such as medical care, environmental testing, and food safety.3,4 The main hardware components of the optical disc technology are disc media and computer drives/disc players for which the recording and reading are managed by software drivers. In adapting the circular geometry and the mechanical operation (spinning) of optical discs, “lab-on-a-disk” (LOD) devices (centrifugal microfluidic platforms) that can perform automatic manipulation of liquid samples by exploiting centrifugal force and the capillary force of microfluidic channels have remained as a major focus.5,6 Other researchers have modified the computer drives as universal optical signal reading devices, as the optical pick-up unit contains both a light source (the laser) and a detector (the photodiode).7– 10

In the meantime, the concept of optical disc has been adapted to the development of bioCD11

and bioDVD12 as label-free detection platforms for biomolecular recognition, and the disc media have been investigated as substrate materials for preparing bioassays that can be read with advanced spectroscopic techniques (surface Plasmon resonance,13,14 surface enhanced Raman spectroscopy15,16, as examples). Optical disc technology has witnessed significant changes in their market share since the commercialization of USB flash drives in 2000;17 as of today Blu-ray discs (BDs) are most likely the survivors as they are popularly used to store high-definition movies and 3D games. Blu-ray technology is more advantageous than the compact disc (CD) and digital versatile disc (DVD) recording; besides improved fabrication of the disc media (vide infra) the drive utilizes a laser with a shorter-wavelength (405 nm vs. 650 nm for DVD and 780 nm for CD, respectively) and an objective lens with larger numerical aperture (NA = 0.85 vs. 0.6 for DVD and 0.45 for CD, respectively).18 The improved optics results in a much smaller laser focusing spot, therefore significantly increases the storage capacity (25 GB vs. 4.7 GB for DVD). Nevertheless, only limited studies have been reported in exploring BD technology for the development of analytical devices.19 Arnadis-Chover et al. has modified a Blu-ray drive by incorporating an external amplification/detection board for reading disc-based high-density DNA hybridization arrays and competitive immunoassays for microcystins.20 Nieuwoudt et al. sputtered gold films on the nanostructured gratings of a BD to create inexpensive substrates for surface-enhanced Raman spectroscopy.21 Donolato et al. ACS Paragon Plus 3 Environment


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converted a Blu-ray optical pick-up unit into an optomagnetic reading device to monitor the Brownian relaxation dynamics of modified magnetic nanoparticles in microfluidic devices for the quantitation of E. coli DNA.22 Independently, Tian et al. reported Blu-ray technology-based optomagnetic reading of a competitive immunoassay for Salmonella.23 We have been interested in the development of digitized molecular detection systems by using unmodified optical drives to read various chemical and biochemical assays prepared on bona fide optical discs (CDs and DVDs)3. Such a conceptually different approach has been validated and improved by others for cell counting24 and for the detection of food allergens.25 Herein we explore the possibility of creating a Blu-ray technology-based analytical platform with unprecedented sensitivity and resolution; beyond the search of new activation methods that work for the special coatings on BD discs, we need to identify an assay system that has practical applications either for on-site chemical analysis or for point-of-care medical diagnosis. For the latter, we have decided to tackle the detection of cardiac markers, as their on-time and accurate detection is crucial for acute myocardial infarction (AMI) diagnosis. Cardiovascular diseases (CVD) are the leading cause of death worldwide;26,27 according to the newest data reported from the World Health Organization and American Heart Association (AHA), globally 8.1 million people died from AMI, accounting for 1 in every 7 deaths worldwide in 2013.28,29 Currently, AMI is diagnosed predominantly from any two combinations of determined AMI-relevant chest pain, related findings from electrocardiographic (ECG) examinations30,31 and increased concentration of cardiac biomarkers, particularly myoglobin (MB) and cardiac troponin I or cardiac troponin T (cTnI or cTnI, respectively), and C-creative protein (CRP). During AMI, the concentration of the respective protein markers increases significantly with time.30,31 The best solution is to test several cardiac biomarkers simultaneously to improve accuracy, sensitivity and specificity for the diagnosis of AMI and risk of reinfarction.

2. Results and discussion 2.1. Digital reading protocol for “ink assays” on a BD The storage capacity of a BD-R is 25 GB; we have recorded high-definition (HD) video files that typically have an average size of 8 GB for movies of 2 hours length for testing the assay reading protocol described below. When reading the disc with recorded video files, errors may occur due to the noise arising from disc tilting, laser defocusing during the burning process or even from scratches, dust, and fingerprints left on the disc surface. Since the BD reading system is very sensitive to small particles/minor scratches on the surface, the structure of BD error correction code (ECC) cluster has been improved by inserting picket columns between the user data in order ACS Paragon Plus 4 Environment

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to provide better error correction capability. LDC (long distance code) and BIS (burst indicator subcode) are the two error testing modes used in the BD-quality diagnostic software to construct error distribution histograms and indicate the number of reading errors exist. To start with we examined the disc after the recording process by using the free PlexUTILITIES 1.3.3.1 program. This software can determine the LDC and BIS error counts for every 0.0625 megabyte (MB) of data, which is the size of an ECC (error correction code) block in the Blu-ray system. Particularly, we have drawn 21 ink stains of different sizes using a Pilot extrafine metallic ink marker on a BD disc with previously recorded data (Figure 1a). As shown in Figure 1b, the BIS error reading yielded 21 distinct peaks with varied heights and widths. The highlighted four peaks correspond to the dots magnified in the inset of Figure 1(a). Generally, the peak sizes correlate with the dimensions of the ink dots which have diameters from 96 to 605 μm. The smallest ink stain (< 100 μm in diameter) located at the far left is almost invisible in the photo; rather remarkably, the error reading program can produce a distinct peak. This result confirms that a BD has a much better lateral resolution for identifying “ink assays” than a CD (~250 μm)32 or a DVD (~180 μm).33 To physically locate each of the ink stains, we plotted the radial distances of the ink spots (measured from the center of the disc to the center of each stain) as a function of the logical position of the error peak. As depicted in Figure 1(c), the correlation between the two (radial distance and logical position) can be fitted to the following equation: r =

.

× 5.8 − 2.4

+ 2.4

(1)

where r is the radial distance on a BD (cm) and x is the corresponding logical position (GB). A single layered BD-R has the capacity of 23.3 GB and its outer and inner radius are 5.8 cm and 2.4 cm, respectively. With this equation, one can readily determine the physical location of an assay site providing the logical position (with the pre-recorded data) is known. For example, the last error peak located at the logical position of 21.6 GB is produced by the ink stain located at 5.62 cm from the center of the disc.

2.2. Surface activation and preparation of assay-on-a-BD Since there is a special hard coating material on BD-Rs, we need to explore different surface activation methods other than the UV/ozone treatment that was successful for the CD/DVD surface (polycarbonate substrate).32,33 The hard coating material on BD-Rd is typically an UVcurable resin with dispersed silica nanoparticles.34 Before performing any surface reaction, we examined the hard coating material of BD-Rs using energy-dispersive X-ray spectroscopy (EDS)



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and Infrared spectroscopy (IR), and confirmed that the featured Hard Coat™ of Verbatim BD-Rs is, in fact, based on lactone composites.35 We have used a common base, sodium hydroxide (0.1 M), to perform a hydrolysis reaction (90 min) on BDs at an elevated temperature (55 °C); the surface was characterized by measuring the value of contact angles using a set of buffer solutions (pH = 2 – 12). As examined with atomic force microscopy (AFM), no corrosion or significant changes to the surface morphology was observed after the activation. In other words, the treated BDs are still readable by an unmodified optical drive. As shown in Figure 2(a), the contact angle remains at 98.9 ± 1.6°on the untreated surface throughout; in contrast the contact angles of the hydrolyzed BD-R surface show a clear transition from 78 ± 2° to 51 ± 2° at pH 4 to 9. This transition in surface hydrophilicity indicates that reactive carboxylic acid groups were generated on the surface. The extracted pKa (~6) is comparable with that obtained on carboxy-terminated self-assembled monolayers on gold.36 It has been observed previously that pKa value on the surface is typically 2-4 pH units higher than that of the solution pKa (3-5).37 Such a difference reflects the increased difficulty of generating the charged form of functional groups (from –COOH to –COO-) at the solid/liquid interface.38 The surface density of thus generated carboxylic acid groups was determined to be 1.1 ± 0.1 × 10-8 mol/cm2 on a BD surface, which is much higher than that on the surface of CDs/DVDs (4.8 ± 0.2 × 10-10 mol/cm2).39 The challenging question is what exact reactions have occurred on the surface upon hydrolysis; in Figure 2(b) we listed three possible products (A and B) that could be formed in the (meth)acrylate-based lactone coating.40 The first product comes from the hydrolysis of ester groups on the side chain, while another results from opening of the lactone ring via acyl-oxygen cleavage.41,42 To identify which ones are most favorable, several factors such as polymer structure and temperature need to be considered, which is beyond the scope of this paper. Most importantly, the generation of reactive carboxy groups on the BD surface provides a viable means to covalently attach probe molecules (via amide-coupling) for preparing the bioassays of interest. Maquieira and co-workers have explored the preparation of bioassays on the BD surface based on physioadsorption, i.e., either streptavidin (for immobilizing biotinylated DNA strands) or a coating conjugate (BSA-MC, for the detection of microcystin LR) was spotted on the surface using a contact liquid dispenser or a non-contact arrayer.20 Later on, they have improved the immobilization strategy by spin-coating a thin layer of SU-8 to which thiolated oligonucleotides were attached via a thiol-epoxy coupling reaction.43 In this study we directly immobilize the capture antibodies on the BD surface via amide-coupling upon the activation of the surface carboxy groups with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).


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As shown in Figure 3, the analyte was introduced to the assay surface after the successful probe antibody immobilization. Different from the standard immunoassays for MB and cTnI (see Supporting Information), we have prepared an aptamer-antibody hybrid assay for the detection of CRP. The CRP-specific aptamer (a 72-mer oligonucleotide) has been previously selected and characterized by Huang et al.44 The 5’-end of this aptamer is labeled with biotin; the introduction of nanogold-streptavidin conjugates facilitates the following signal enhancement step (i.e., silver staining). Since biomolecules are too small (

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