A compact disk/digital versatile disk (CD/DVD) based immunoassay is a method for determining the concentration of a compound in research and diagnostic laboratories by performing the test on an adapted CD/DVD surface using an adapted optical disc drive; these methods have been discussed and prototyped in research labs since 1991. [1]
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 can act 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. [1]
Because the disk spins, the platform has the ability to drive the sample through it through microfluidic channels and for multiple steps to be performed, allowing the possibility for sample preparation and more than one analysis to be conducted during a single run. [1]
CD/DVD based assays could potentially be used for any immunoassay already in use and many assays used in analytical chemistry, as long as analytes have a corresponding probe, are soluble, and are large enough to alter the angle of incident. [1]
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 can be activated to reveal the metal layer which allows compounds to bind to it. Compounds such as UV/ozone or an oxygen plasma treatment can be used to activate the disk to produce a hydrophilic surface with densely packed carboxylic acid groups. [1]
As one-off microassay can be printed onto the activated disks using a noncontact printer to dispel nanoliter quantities of coating conjugates onto the disk. Proteins or antibodies acting as probe molecules can then covalently bind to the disk surface and can be 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 can bind or hybridize at the intersections of the arrays to create rectangular hybridization sites. The disk is washed, rinsed, and dried prior to reading. [1] This process can be done manually or automated; in theory discs with pre-made assays could be manufactured and sold en masse. [1]
Standard CD/DVD readers can be used to read the assays. The CD/DVD readers contain a laser, set of optical elements which shape and focus the laser, a disk driver, and a signal detector that function as follows: [1]
The signal or optical density of the image is inversely proportional to concentration. [1] 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. [3]
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. [4]
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. [5] The gold nanoparticles can be identified with photometric or plasmonic detectors. The smaller the nanoparticles are, the more sensitive the assay becomes.
Silver enhancer solution is also used to increase the reflective properties of samples. 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 additional reaction and washing steps which could lead to analytical errors. [1]
As of 2016, challenges including optimizing sample treatment, optimizing disk surfaces, developing readers that can deploy multiple colors of light delivery and sensing for multiplexing, and for clinical use, obtaining regulatory approvals. The field is similar to lab-on-a-chip platforms. [1]
As of 2010 companies including Gyros AB, Tecan, and Burstein Technologies were working on bringing CD/DVD based immunoassays and equipment to market. [6] [7]
The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemistry assay, first described by Engvall and Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.
Digital microfluidics (DMF) is a platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets are dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.
A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector. The sensitive biological element, e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc., is a biologically derived material or biomimetic component that interacts with, binds with, or recognizes the analyte under study. The biologically sensitive elements can also be created by biological engineering. The transducer or the detector element, which transforms one signal into another one, works in a physicochemical way: optical, piezoelectric, electrochemical, electrochemiluminescence etc., resulting from the interaction of the analyte with the biological element, to easily measure and quantify. The biosensor reader device connects with the associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. This sometimes accounts for the most expensive part of the sensor device, however it is possible to generate a user friendly display that includes transducer and sensitive element. The readers are usually custom-designed and manufactured to suit the different working principles of biosensors.
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The analyte can be a drug, biochemical substance, chemical element or compound, or cell in an organism or organic sample. The measured entity is often called the analyte, the measurand, or the target of the assay. An assay usually aims to measure an analyte's intensive property and express it in the relevant measurement unit.
Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.
An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes.
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of biomolecules and various organic molecules, which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to electrospray ionization (ESI) in that both techniques are relatively soft ways of obtaining ions of large molecules in the gas phase, though MALDI typically produces far fewer multi-charged ions.
In analytical chemistry, the detection limit, lower limit of detection, or LOD, often mistakenly confused with the analytical sensitivity, is the lowest quantity of a substance that can be distinguished from the absence of that substance with a stated confidence level. The detection limit is estimated from the mean of the blank, the standard deviation of the blank, the slope of the calibration plot and a defined confidence factor. Another consideration that affects the detection limit is the accuracy of the model used to predict concentration from the raw analytical signal.
Heterophile antibodies are antibodies induced by external antigens.
An antibody microarray is a specific form of protein microarray. In this technology, a collection of capture antibodies are spotted and fixed on a solid surface such as glass, plastic, membrane, or silicon chip, and the interaction between the antibody and its target antigen is detected. Antibody microarrays are often used for detecting protein expression from various biofluids including serum, plasma and cell or tissue lysates. Antibody arrays may be used for both basic research and medical and diagnostic applications.
Surface-enhanced laser desorption/ionization (SELDI) is a soft ionization method in mass spectrometry (MS) used for the analysis of protein mixtures. It is a variation of matrix-assisted laser desorption/ionization (MALDI). In MALDI, the sample is mixed with a matrix material and applied to a metal plate before irradiation by a laser, whereas in SELDI, proteins of interest in a sample become bound to a surface before MS analysis. The sample surface is a key component in the purification, desorption, and ionization of the sample. SELDI is typically used with time-of-flight (TOF) mass spectrometers and is used to detect proteins in tissue samples, blood, urine, or other clinical samples, however, SELDI technology can potentially be used in any application by simply modifying the sample surface.
A molecular sensor or chemosensor is a molecular structure that is used for sensing of an analyte to produce a detectable change or a signal. The action of a chemosensor, relies on an interaction occurring at the molecular level, usually involves the continuous monitoring of the activity of a chemical species in a given matrix such as solution, air, blood, tissue, waste effluents, drinking water, etc. The application of chemosensors is referred to as chemosensing, which is a form of molecular recognition. All chemosensors are designed to contain a signalling moiety and a recognition moiety, that is connected either directly to each other or through a some kind of connector or a spacer. The signalling is often optically based electromagnetic radiation, giving rise to changes in either the ultraviolet and visible absorption or the emission properties of the sensors. Chemosensors may also be electrochemically based. Small molecule sensors are related to chemosensors. These are traditionally, however, considered as being structurally simple molecules and reflect the need to form chelating molecules for complexing ions in analytical chemistry. Chemosensors are synthetic analogues of biosensors, the difference being that biosensors incorporate biological receptors such as antibodies, aptamers or large biopolymers.
Lateral flow tests (LFTs), also known as lateral flow immunochromatographic assays or rapid tests, are simple devices intended to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment. These tests are widely used in medical diagnostics for home testing, point of care testing, or laboratory use. For instance, the home pregnancy test is an LFT that detects a certain hormone. These tests are simple, economic and generally show results in around five to 30 minutes. Many lab-based applications increase the sensitivity of simple LFTs by employing additional dedicated equipment.
The centrifugal micro-fluidic biochip or centrifugal micro-fluidic biodisk is a type of lab-on-a-chip technology, also known as lab-on-a-disc, that can be used to integrate processes such as separating, mixing, reaction and detecting molecules of nano-size in a single piece of platform, including a compact disk or DVD. This type of micro-fluidic biochip is based upon the principle of microfluidics; to take advantage of noninertial pumping for lab-on-a-chip devices using noninertial valves and switches under centrifugal force and Coriolis effect to distribute fluids about the disks in a highly parallel order.
Mass spectrometric immunoassay (MSIA) is a rapid method is used to detect and/ or quantify antigens and or antibody analytes. This method uses an analyte affinity isolation to extract targeted molecules and internal standards from biological fluid in preparation for matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). This method allows for "top down" and "bottom up" analysis. This sensitive method allows for a new and improved process for detecting multiple antigens and antibodies in a single assay. This assay is also capable of distinguishing mass shifted forms of the same molecule via a panantibody, as well as distinguish point mutations in proteins. Each specific form is detected uniquely based on their characteristic molecular mass. MSIA has dual specificity because of the antibody-antigen reaction coupled with the power of a mass spectrometer.
Lanthanide probes are a non-invasive analytical tool commonly used for biological and chemical applications. Lanthanides are metal ions which have their 4f energy level filled and generally refer to elements cerium to lutetium in the periodic table. The fluorescence of lanthanide salts is weak because the energy absorption of the metallic ion is low; hence chelated complexes of lanthanides are most commonly used. The term chelate derives from the Greek word for “claw,” and is applied to name ligands, which attach to a metal ion with two or more donor atoms through dative bonds. The fluorescence is most intense when the metal ion has the oxidation state of 3+. Not all lanthanide metals can be used and the most common are: Sm(III), Eu(III), Tb(III), and Dy(III).
Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps.
Multiplexed point-of-care testing (xPOCT) is a more complex form of point-of-care testing (POCT), or bedside testing. Point-of-care testing is designed to provide diagnostic tests at or near the time and place that the patient is admitted. POCT uses the concentrations of analytes to provide the user with information on the physiological state of the patient. An analyte is a substance, chemical or biological, that is being analyzed using a certain instrument. While point-of-care testing is the quantification of one analyte from one in vitro sample, multiplexed point-of-care testing is the simultaneous on-site quantification of various analytes from a single sample.
A kinetic exclusion assay (KinExA) is a type of bioassay in which a solution containing receptor, ligand, and receptor-ligand complex is briefly exposed to additional ligand immobilized on a solid phase.
A chemical sensor array is a sensor architecture with multiple sensor components that create a pattern for analyte detection from the additive responses of individual sensor components. There exist several types of chemical sensor arrays including electronic, optical, acoustic wave, and potentiometric devices, which are described below. These chemical sensor arrays can employ multiple sensor types that are cross-reactive or tuned to sense specific analytes.