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WIREs Nanomed Nanobiotechnol
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ELISA‐type assays of trace biomarkers using microfluidic methods

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Recently, great progress has been achieved for analytical technologies for biological substances. Traditionally, detection methods for analytes mainly rely on large instrumental analyses. These methods require costly equipment, skilled operators and long measurement time despite their generally low sensitivity. In contrast, immunoassays are becoming more and more popular for it is powerful, inexpensive, and convenient nature. Immunoassay has a range of applications, because it employs antibody, a protein produced by plasma cells in the acquired immune system to identify and neutralize diverse pathogens and other exogenous substances. However, the sensitivity of conventional immunoassays so far is limited by their reaction principles and detection methods. The microfluidics technology is the one that manipulates small volumes of fluid and flow, which has the potential to miniaturize many laboratory procedures. Immunoassays on microfluidic devices have been studied extensively and have gained significant attention owing to intrinsic advantages offered by the assay platforms. The techniques have allowed the miniaturization of conventional immunoassay and bring the advantages such as small volumes of samples and reagents as well as the decrease of contamination, which results in the decline of false‐positive results. Ultimately, the combination of immunoassays with microfluidics affords a promising platform for multiple, sensitive, and automatic point‐of‐care diagnostics. Recent achievements on microfluidic devices and immunoassay detection systems including digital assay employing single molecule will be introduced in detail and the strategies for faster and more sensitive configurations in microfluidic immunosensors will be highlighted. WIREs Nanomed Nanobiotechnol 2017, 9:e1457. doi: 10.1002/wnan.1457 This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices
General principles of immunoassays. (a) Sandwich enzyme‐linked immunosorbent assay (ELISA), (b) competitive ELISA, and (c) open‐sandwich ELISA. Ab, antibody; Ag, antigen; VH, variable region of antibody heavy chain; VL, variable region of antibody light chain. [Correction added on 27 April 2017 after first online publication: following original publication, Figure 1 has been amended to improve visibility of Y shape antibodies.]
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Conceptual schematic of a digital measurement using Brownian trapping with drift. This concept aims to combine the ultrasensitivity of digital detection (shown in blue) with large changes in the input concentration raised by Brownian trapping (shown in red).
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Microfluidic device used for femtodroplet generation and manipulation. (a) Femtodroplet formation at the nozzle of the microfluidic device. (b) Photograph of the whole multilayered polydimethylsiloxane (PDMS) device. The upper layer consists of the nozzle, flow channels, and storage compartments, with a capacity for <2 × 105 femtodroplets. The bottom layer houses the monolithic valves used to control droplet flow and isolate the traps. (c) After the on‐chip incubation, three populations of femtodroplets are observed: (i) droplets containing no bead, (ii) those containing a bead without immunocomplexes, and (iii) those containing a bead with an immunocomplex exhibiting a positive fluorescence signal due to the enzymatic activity of a single β‐galactosidase reporter. The numerical ratio of (iii) to [(ii) + (iii)] yields the concentration of the target analyte.
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Digital enzyme‐linked immunosorbent assay (ELISA) based on arrays of femtoliter‐sized wells. (a) Single protein molecules are captured and labeled on beads using standard ELISA reagents and (b) beads with or without a labeled immunoconjugate are loaded into femtoliter‐volume well arrays for isolation and detection of single molecules by fluorescence imaging.
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Digital enzyme‐linked immunosorbent assay (ELISA) based on the detection of single immunocomplexes in arrays of femtoliter wells. Step A: capturing of single protein molecules on paramagnetic beads coated in capture antibodies; Step B: labeling of capture proteins with a biotinylated detection antibody and then with an enzyme conjugate; and Step C: loading of beads suspended in enzyme substrate into arrays of femtoliter‐sized wells for isolation and detection of single molecules.
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Microfabricated arrays of femtoliter chambers allow single‐molecule detection. (a) Microfabrication of the polydimethylsiloxane (PDMS) chambers. (A) Fabrication of the mold: a 1.5‐m‐thick Silicon on Insulator (SOI) wafer was covered with an aluminum mask, itself patterned by photolithography. After etching and removing the aluminum layer, the silicon surface showed regular arrays of identical cylindrical shapes. (B) Scanning electron microscopy (SEM) image of the silicon mold in a region patterned with 5‐m microstructures. (C) After Teflon coating of the mold, liquid PDMS was poured, cured at 90°C for polymerization, and peeled off. (D and E) SEM images of the PDMS sheets: mold patterns were precisely reproduced at both the 5‐m (D) and the 1‐m (E) level, for which the chambers had an internal volume of 1.4 fL. (b) Water enclosure in the microchambers and evaluation of the sealing. (A) Sealing process: the buffer solution that contains the molecules of interest is sandwiched between a glass plate and the PDMS layer. Upon pressure, PDMS attached tightly to the glass surface and closed the chambers, impeding diffusion. (B–D) Inclusion of plastic beads (B), quantum dots (C), and DNA chains (D) in variously sized microchambers. (E) Photobleaching experiment using sulforhodamine G (see F). (G) A caged fluorescein compound (see F) can be activated selectively inside a few chambers using a focused UV‐light beam.
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Micro open‐sandwich enzyme‐linked immunosorbent assay (OS‐ELISA) system for detection of osteocalcin. (a) Micro OS‐ELISA system. A glass chip (3 cm × 7 cm) integrated with microfluidic channels (200 µm × 200 µm) was used for the OS‐ELISA reaction. Polystyrene beads with immobilized maltose‐binding protein (MBP)‐VL were introduced from the inlet (connector) and stored between a dam structure and the three‐way junction, and extra beads were flushed through the stop valve. MBP‐VH‐HRP (horseradish peroxidase) and antigen on the rotating stage were introduced into the microchip to form an immuno‐complex on the beads. After washing, the substrates were applied and dye molecules produced by the enzyme reaction were detected by a thermal lens microscope (TLM) downstream of the dam structure. The microbeads after each assay were flushed by an inversed flow. (b) Scheme of immunoassay reaction and detection of signal.
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Conceptual schematic of the integrated microfluidic aqueous two‐phase extraction (ATPE) strategy for matrix neutralization and ochratoxin A (OTA) concentration in red wine samples. SRW, red wine spiked with salt; PRP, PEG‐rich phase.
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A microfluidic indirect competitive immunoassay for detection of testosterone. (a) The illustration of indirect competitive reaction for assaying testosterone. (b) Schematic of microfluidic assay for detection of testosterone. (i, ii) Introduction and immobilization of antigen testosterone–bovine serum albumin (BSA) onto the polydimethylsiloxane (PDMS) substrate via a top PDMS layer; (iii) block the nonspecific binding sites with 3% BSA; (iv) indirect competitive reaction by introduction of the testosterone sample and anti‐testosterone monoclonal antibody via a top PDMS layer; (v) introduction of the secondary antibody IgG–horseradish peroxidase (HRP); and (vi) introduction of the chemiluminescent substrate to obtain the signal spots of immunoassays.
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Microchip‐based enzyme‐linked immunosorbent assay (microELISA) for detection of biomarker. (a) Assay scheme and (b) dam structure fabricated in a microchip.
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A magnetic bead‐based microfluidic platform for detection of oligomer amyloid‐β assay in a microfluidic device. (a) Schematic drawing of the multimer detection system. Anti‐Aβ monoclonal antibodies were used for both capture and detection. Monomers are not able to bind both capture and detect antibodies, but oligomers bind to both antibodies because of the presence of two or more epitopes. Horseradish peroxidase (HRP) conjugated on detection antibody catalyzes substrate to generate a fluorescence signal. (b) Structure of chamber. Chamber (i) is for the incubation of antigen, capture antibody‐conjugated magnetic bead, and HRP‐conjugated detection antibody. Chamber (ii) is for washing. Chambers (iii) and (iv) contain mineral oil for separating reagents. Chamber (v) is for the detection of signal.
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