Array-on-a-disk? How Blu-ray technology can be applied to molecular diagnostics

Abstract

This editorial comments on the balance and perspectives of compact disk technology applied to molecular diagnostics. The development of sensitive, rapid and multiplex assays using Blu-ray technology for the determination of biomarkers, drug allergens, pathogens and detection of infections would have a direct impact on diagnostics. Effective tests for use in clinical, environmental and food applications require versatile and low-cost platforms as well as cost-effective detectors. Blu-ray technology accomplishes those requirements and advances on the concept of high density arrays for massive screening to achieve the demands of point of care or in situ analysis.

Keywords::

• biosensing
• Blu-ray technology
• compact disk
• massive screening
• point-of-care diagnostics

Since compact disc (CD) technology came out, billions of digital compact discs and players have been used as a comparatively inexpensive form of portable data storage and reading system.

Despite the essential physical differences between CDs, DVDs and Blu-ray discs (BDs) [1], the basic structure comprises a grooved polycarbonate substrate with encoded digital information covered by a nanometer thin metallic (Ag or Al) film to provide high reflectivity to the disc. Also, a protective lacquer, polymer or a hard coating resin, for CDs, DVDs and BDs, respectively, constitutes the finishing material. These digital discs have outstanding physical properties such as impact resistance, heat stability, large surface and good protein adsorption efficiency.

The current analytical applications using discs as supports can be categorized into two groups. One uses plastic CD-like surfaces without data storage capacities on which microfluidic structures are integrated to facilitate the analysis development. Examples of this strategy have ended up in point-of-care industrial devices offered by Gyros (Gyrolab), Quanterix (Simoa) and Pall (GeneDisc) that require large and sophisticated benchtop equipment for the analysis and detection. The second strategy exploits the basic reading principle and components of the audio–video technology for developing the assays. In this sense, the principle of CD-based microarrays was first reported in 2000 in a work that successfully combined low-density microarrays applied via a piezoelectric inkjet printer with circular indexing on a polycarbonate disc [2]. This seminal work concluded with the amazing idea the simpler the better using disc-based microarrays without both microfluidics and expensive detection devices such as fluorescence scanners or microscopes.

From that work to now, several fundamental contributions have played an important part in the crystallization of this remarkable sensing concept. Indeed, the potential of optical disc drive as the signal-transducing element has been demonstrated for quantitative analysis using regular CDs and DVDs [3–5]. Discs have been used as analytical platforms to immobilize probes (proteins and nucleic acids) directly by passive adsorption [6,7], indirectly by means of affinity bioreagents [8] (streptavidin, protein A, concanavalin A, antibodies, etc.), covalently by previous chemical functionalization of the original disc polymer (polycarbonate) [9] or by spin coating a supplementary film such as polystyrene [10], SU-8 [11], gold, aluminum oxide or graphite [12]. These strategies were developed to successfully attach probes performing different analytical applications. The technology has demonstrated potential for multiplex screening analysis, determining antibiotics, pesticides, aquatic toxins with detection limits below 0.5 µg/l, biomarkers below the basal level, food-borne pathogens within 10⁰–10¹ cfu/ml and food allergens below 1 μg/g (0.0001%). The assay time goes from 25 to 60 min depending on the application. These features make CD technology suitable for screening purposes within the environmental monitoring, food/water safety and diagnostic tests, reaching similar sensitivity and selectivity to the ELISA and qPCR assays.

To advance on the development of novel molecular diagnostics using CD technology-based systems, the use of the newest generation of mass-produced commercial optical discs such as Blu-ray is interesting. The structure of a BD disc consists on a 1.1 mm grooved polycarbonate substrate metalized with nanometer reflective film of aluminum alloy (40–100 nm thick). This film is protected with a 98 and 2 μm thick light transmitting and hardcoat layers, respectively [13]. In BD system, the use of short wavelength laser diodes (405 nm) and high numerical aperture objective lens makes it possible to achieve recording and detection densities in microarray format. For this, the blue laser beam enters the bottom side of the disc with a spot diameter of 130 µm due to the reflective index, and the thickness of the polycarbonate substrate is focused on the metalized layer to 0.42 µm [1]. The thickness and transparency of the light transmitting layer is critical to maintain laser beam focused on the recording medium while the disk is rotated (800–2000 r.p.m.) to produce microstructural changes (pits) in the recording layer. The laser beam is reflected back with the micro pattern of the pits to a photodiode integrated into an optical pickup assembly. This signal is detected and converted into analog information. The electronic circuitry and the precise focusing optics permit the development of robust, integrated and high performance, low-cost optical systems.

The composition of the hardcoat layer of BDs is also an interesting issue to molecular diagnostics. The layer is made of colloidal silica-dispersed UV-curable resin, chemically inert and highly hydrophobic, scratch-resistant antistatic material that allows adsorption of probes. This feature makes BD an exceptional planar surface for molecular diagnostics as recently demonstrated [14]. In this pioneer work, the bottom side (hard-coat) of the BD is modified by coating it with protein probes, by passive adsorption, in a high-density format (64–100 spots/mm²) achieving an immobilization density of 2.0 fmol/mm². To reach such high probe density, contact printers were used to dispense few picoliters of solutions, generating spots of 85 µm in diameter. Once the molecular assay is performed, an enzymatic signal amplification step is carried out to generate an insoluble reaction product that modifies the reflection properties of the disk, reducing the signal intensity measured by the photodiode. The ordinary Blu-ray drive quantifies the change in the reflective properties of the disk, but in our case, the interference (attenuation of the reflected signal) correlates well with the concentration, optical density or other properties of the bioreaction product. The described readout system requires a physical access to the optical disk drive to obtain the analog signal that is converted into an image. This data acquisition process detect small changes in the analog signal, resulting in sensitive quantitative measurements with high dynamic range. This approach has been applied to the detection of food-borne pathogens (Salmonella spp. and Cronobacter spp.) and for the determination of aquatic toxins displaying similar or better results in terms of sensitivity and reproducibility to DVD and qPCR technologies. It is interesting to note that this approach combined in the same platform immune and nucleic acid-based assays, broadening the analytical perspectives of the technology. In addition, the use of shorter wavelength, compared with CD and DVD technology, ensures that the laser beam is focused on the polycarbonate surface with a smaller size; thus, the detection method would be more sensitive with a better lateral resolution, increasing the multiplex throughput as well.

Recently, the capability of spin coating SU-8 material to covalently attach oligonucleotides on a BD has also been reported [10]. This strategy opens the way to functionalize BD surface in a simple manner for sensing molecular diagnostics applications, preserving the optical and mechanical properties of the original disk.

In spite of the considerable advance in the use of optical disk technology for molecular diagnostics, still some interesting challenges do exist. The first concerns to the simultaneous multiwavelength operation. A conventional BD drive can read all the available optical disks such as CDs and DVDs, but only one laser beam is turned on during disk scanning, misusing the optical capacity of the device. New hardware developments would permit the simultaneous use of the lasers, reaching similar or better performances as those of fluorescence scanners in terms of optical resolution and scanning time. Also, the use of partially transparent BDs can be an interesting approach due to the better optical resolution capabilities. Similar attempts have been already described for CDs and DVDs with remarkable results [15]. This would allow scanning nanometric structures down to 420 nm; this in combination with the rapidity to scan a large sensing would make Blu-ray technology suitable for high-density mass screening applications.

Another challenging approach is related with the implementation of label-free assay-based formats as it has been already reported for other type of discs [16]. In this line, interferometry as detection principle using rewritable BDs is an alternative to give the best results. This challenge would involve the implementation of an interferometer into the conventional drive without losing its capacity to scan regular BDs.

Molecular diagnostics is an intensive analytical field that develops rapidly and demands mass-produced technologies with bright performances as sensitivity, portability, ubiquity, reliability and low cost. Blu-ray technology fulfills these requirements and provides additional advantages such as interesting surface functionalities and in situ analysis detection what make this system suitable for massive screening purposes based on molecular recognition and spatial resolution principles.

Acknowledgements

This research was partially supported by projects GVA-PROMETEO/2010/008 (Generalitat Valenciana), CTQ/2010/15943 (MICINN) and CTQ2013-45875-R (MINECO).

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.