CD/DVD based immunoassay

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Illustration of the basic componenets of a CD/DVD based immunoassay, which includes the disk (grey), a probe (green), gold nanoparticle (red), analyte (yellow), and silver particle (blue)

A compact disk/digital versatile disk (CD/DVD) based immunoassay is a biochemical analytical test which determines the concentration of a compound by performing the test on a standard CD/DVD surface. Samples are bound to the surface of the CD or DVD in a microarray format, and an antibody binds to the compound of interest. The CD/DVD based assays are read using standard CD/DVD readers. The technique using CDs/DVDs is cheaper than many comparable techniques.[1]

Principle

CDs and DVDs have a polycarbonate surface and metal reflective layer which allow for storage and retrieval of information. The metal film is sometimes made of pure gold which is highly stable and has ideal optical properties.[2] The metal acts as a substrate which allows compounds to bind to it. This alters the reflective and refractive properties of the disk. Disk reading is based on capturing analog signals with the disk drive. The signals are indicative of how much analyte is in a sample.

Applications

The technique has a wide range of applications. CD/DVD based assays can be used as long as analytes have a corresponding probe, are soluble, and are large enough to alter the angle of incident light.

Some uses include:

Microarray Platform

Microarray

CDs and DVDs have a protective film which must be stripped to reveal the gold reflective film or polycarbonate (PC) base. The surface of the disk is activated to reveal the metal layer which allows compounds to bind to it. Compounds such as UV/ozone or an oxygen plasma treatment are used to activate the disk to produce a hydrophilic surface with densely packed carboxylic acid groups.[10][11] The microassays are printed onto the activated disks using a noncontact printer to dispel nanoliter quantities of coating conjugates onto the disk.[3][4][5][7][10][11] Proteins or antibodies acting as probe molecules covalently bind to the disk surface and are incubated. A polydimethylsiloxane (PDMS) channel plate can also be used to immobilize the probes in a line array. The plate is removed, and the process is repeated with another plate to deliver analyte samples in a line array perpendicular to the probe array. The probe and analyte samples bind or hybridize at the intersections of the arrays to create rectangular hybridization sites. The disk is washed, rinsed, and dried prior to reading. The manual creation process is slower than using a printer. A noncontact printer loaded with probe, sample, buffer, and wash solution allows the process to be completely automated.[6]


Instrumental Analysis

Standard DVD reader

Standard CD/DVD readers can be used to read the assays.[3][4][10] The CD/DVD readers contain a laser, set of optical elements which shape and focus the laser, a disk driver, and a signal detector.[8]

  1. The laser produces light of a selected wavelength.
  2. The beam of light hits the analyte in the spots of the microarrays and refracts. The mass of the analyte causes the angle of reflected light to be different from the angle of incident light. The reflective properties of the CD/DVD change based on the quantity of analyte in the sample.
  3. The attenuated signal reaches the photodiode of the drive's pickup.
  4. Analog signals are extracted, digitized, and converted to an image.

The signal or optical density of the image is inversely proportional to concentration.[11] The refractive index of light, which is directly proportional to concentration, can also be measured. A readable signal is only generated if the sample is at least 200 nm, otherwise it is too small to significantly disrupt reflection of incident laser light.[12]

DVD diagnostic software programs such as Kprobe, ODC, and PlexUtilities can also be used for testing arrays and assays prepared on DVDs. These programs rely on a basic DVD error correcting algorithm. DVDs are organized by sectors which each consist of 2064 bytes. A logical error correction code (ECC) block consists of 16 data sectors. The ECC block is the basic unit for testing disk quality by counting the number of parity inner errors (PIE) or parity inner failures (PIF). The software programs can analyze PIF density which is proportional to analyte concentration.[11]

Platform enhancements

If analytes are too small to generate a readable signal for determining concentration, the assay matrix can be modified. CD/DVD based assays utilize the optical properties of gold. Gold nanoparticle bioconjugates are tracers used to increase the sensitivity of the assay.[1][3][4][5][10][11] The gold nanoparticles can be identified with photometric or plasmonic detectors. The smaller the nanoparticles are, the more sensitive the assay becomes.[5]

Silver enhancer solution is also used to increase the reflective properties of samples.[3][4][5][11] Gold nanoparticles have catalytic properties which cause them to reduce silver ions to silver metal. The silver metal deposits on the analytes and causes signals to be amplified. Silver metal is more easily detectable by cameras, scanners, or other drives than is the analyte alone. Still, this enhancement procedure requires many addtional reaction and washing steps which could lead to analytical errors.[5]

Comparison to similar techniques

Standard immunoassays using tags or labels are similar to this technique. Immunoassays are often done in a well-plate and read using a microplate reader. However, analyte concentration can be measured in other ways as well. HPLC is often used to measure concentration. Solid phase extraction (SPE) may be done first to separate compounds before quantification using HPLC.[5] HPLC separates analytes, and they can be detected and quantified by a UV-Vis spectrometer or mass spectrometer coupled to the HPLC. Standard optical density detectors can be used to image assays, whether they are on a disk or not. The concentration of analyte is proportional to the optical density. Determining optical darkness ratios is also comparable in accuracy to analyzing PIF density.[11] Dynamic light scattering (DLS) systems rely on the same principles as standard CD/DVD readers. The angle of backscattered light is measured in both cases.[5]

See also

References

  1. 1 2 3 Morais, Sergi; Tamarit-López, Jesús; Carrascosa, Javier; Puchades, Rosa; Maquieira, Ángel (3 July 2008). "Analytical prospect of compact disk technology in immunosensing". Analytical and Bioanalytical Chemistry 391 (8): 2837–2844. doi:10.1007/s00216-008-2224-4.
  2. 1 2 3 Yu, Hua-Zhong (2004). "New chemistry on old CDs". Chemical Communications (23): 2633–2636. doi:10.1039/B412784F.
  3. 1 2 3 4 5 Avella-Oliver, Miquel; Morais, Sergi; Carrascosa, Javier; Puchades, Rosa; Maquieira, Ángel (16 December 2014). "Total Analysis Systems with Thermochromic Etching Discs Technology". Analytical Chemistry 86 (24): 12037–12046. doi:10.1021/ac502640j.
  4. 1 2 3 4 5 6 7 Dobosz, Paulina; Morais, Sergi; Bonet, Emilio; Puchades, Rosa; Maquieira, Ángel (6 October 2015). "Massive Immuno Multiresidue Screening of Water Pollutants". Analytical Chemistry 87 (19): 9817–9824. doi:10.1021/acs.analchem.5b02354.
  5. 1 2 3 4 5 6 7 8 Dobosz, P.; Morais, S.; Puchades, R.; Maquieira, A. (July 2015). "Nanogold bioconjugates for direct and sensitive multiplexed immunosensing". Biosensors and Bioelectronics 69: 294–300. doi:10.1016/j.bios.2015.03.007.
  6. 1 2 3 Li, Xiaochun; Shi, Maolin; Cui, Caie; Yu, Hua-Zhong (16 September 2014). "Inkjet-Printed Bioassays for Direct Reading with a Multimode DVD/Blu-Ray Optical Drive". Analytical Chemistry 86 (18): 8922–8926. doi:10.1021/ac501870w.
  7. 1 2 Tamarit-López, Jesús; Morais, Sergi; Puchades, Rosa; Maquieira, Ángel (21 December 2011). "Oxygen Plasma Treated Interactive Polycarbonate DNA Microarraying Platform". Bioconjugate Chemistry 22 (12): 2573–2580. doi:10.1021/bc2004268.
  8. 1 2 3 Arnandis-Chover, Tania; Morais, Sergi; González-Martínez, Miguel Ángel; Puchades, Rosa; Maquieira, Ángel (January 2014). "High density MicroArrays on Blu-ray discs for massive screening". Biosensors and Bioelectronics 51: 109–114. doi:10.1016/j.bios.2013.07.045.
  9. ↑ Gubala, Vladimir; Harris, Leanne F.; Ricco, Antonio J.; Tan, Ming X.; Williams, David E. (17 January 2012). "Point of Care Diagnostics: Status and Future". Analytical Chemistry 84 (2): 487–515. doi:10.1021/ac2030199.
  10. 1 2 3 4 Zhang, Lingling; Wong, Jessica X. H.; Li, Xiaochun; Li, Yunchao; Yu, Hua-Zhong (19 May 2015). "Detection and Quantitation of Heavy Metal Ions on Bona Fide DVDs Using DNA Molecular Beacon Probes". Analytical Chemistry 87 (10): 5062–5067. doi:10.1021/acs.analchem.5b00899.
  11. 1 2 3 4 5 6 7 Zhao, Xuejiao; Li, Xiaochun; Cui, Caie; Yu, Hua-Zhong (May 2014). "DVD diagnostic software for reading disc-based bioassays, a comparative study". Sensors and Actuators B: Chemical 195: 116–122. doi:10.1016/j.snb.2013.12.087.
  12. ↑ Yu, Hua-Zhong; Li, Yunchao; Ou, Lily M.-L. (19 February 2013). "Reading Disc-Based Bioassays with Standard Computer Drives". Accounts of Chemical Research 46 (2): 258–268. doi:10.1021/ar300104b.
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