Digital Immunoassay Research Articles

Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR.

This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for "digital" (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12,000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, <500 molecules/mL to >1,000,000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011, DOI: 10.1021/ja2060116), this approach is used to design and test digital RT-PCR devices for quantifying RNA.

Amplifying Immunoassay Sensitivity

Genetic Engineering & Biotechnology NewsVol. 31, No. 18 TutorialAmplifying Immunoassay SensitivityMicrofluidics-Based Microplate Technology Offers Benefits Over Traditional ELISA MethodsPublished Online:14 Oct 2011https://doi.org/10.1089/gen.31.18.10AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byThe Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and MultiplexingSLAS Technology, Vol. 21, No. 4Recent developments in emerging microimmunoassaysBioanalysis, Vol. 5, No. 2A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA)Lab on a Chip, Vol. 12, No. 21 Volume 31Issue 18Oct 2011 Information© 2011 by GEN PublishingTo cite this article:Junhai Kai, Aniruddha Puntambekar, David Sehy, Peter Brescia, Peter Banks.Amplifying Immunoassay Sensitivity.Genetic Engineering & Biotechnology News.Oct 2011.26-27.http://doi.org/10.1089/gen.31.18.10Published in Volume: 31 Issue 18: October 14, 2011PDF download

Nanogel surface coatings for improved single-molecule imaging substrates

Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings. We characterized the nanogel coating to determine its suitability for SM protein experiments. SM adsorption counting revealed that nanogel-coated surfaces exhibit lower protein adsorption than covalently coupled BSA surfaces and monolayers of multi-arm PEG, so this surface displays one of the lowest degrees of protein adsorption yet observed. Additionally, the nanogel coating was resistant to DNA adsorption, underscoring the utility of the coating across a variety of SM experiments. The nanogel coating was found to be compatible with surfactants, whereas the BSA coating was not. Finally, applying the coating to a real-world study, we found that single ligand molecules could be tethered to this surface and detected with high sensitivity and specificity by a digital immunoassay. These results suggest that PEG-BSA nanogel coatings will be highly useful for the SM analysis of proteins.

Development of AccuPSATM, a novel digital immunoassay for subfemtomolar measurement of PSA in postradical prostatectomy patients

Abstract Purpose: Develop a method for measuring prostate specific antigen (PSA) in the serum of patients who have undergone radical prostatectomy (RP). The method is based on Single Molecule Array (SiMoATM) technology in which individual molecules of analyte are trapped in femtoliter microwells and counted. Measurement of PSA in post RP patients 4-6 weeks after surgery (nadir PSA levels) has potential prognostic value. Experimental: The assay starts as a sandwich ELISA except that 2.7 μm paramagnetic beads serve as the solid phase rather than the ELISA plate. The assay is initiated by mixing sample with beads coated with anti-PSA antibody, and the mixture is incubated for two hours. During the incubation, PSA molecules are captured with an excess of beads, such that the ratio of bound PSA per bead is much less than one. Following a wash in which beads are separated using a magnet, biotinylated detector antibody is incubated with the beads for 45 minutes. After a second wash, a conjugate of streptavidin-β-galactosidase is incubated with the beads for 30 minutes to form the final enzyme immunocomplex. Upon completion, the beads are loaded onto the ends of bundles of optical fibers etched with an array of 50,000 microwells, each with a volume of 50 femtoliters (4.5 μm wide × 3.25 μm deep). Single beads are introduced into each well via a 10-minute centrifugation at 1,300 g. Excess beads are removed, and the bundles are sealed against a solution of resorufin β-D-galactopyranoside (RDG). Wells containing an enzyme immunocomplex convert RDG to a fluorescent product, which becomes concentrated in the small volume of the wells. Concentration of the fluorescent product permits ready imaging by a CCD camera and discrimination from wells that contain no enzyme immunocomplex. Poisson statistics predict that each well will contain either one PSA molecule or no PSA molecules when the ratio of bound PSA per bead is much less than one. This results in digitized quantification. Raw signal is recorded as “% active wells”, which can be converted to “average enzymes/bead” to correct for non-Poisson behavior at higher PSA concentrations. The output is related to a standard curve and converted to a PSA concentration of the sample. Results: In preliminary validation studies across multiple runs and reagent batches, the AccuPSA assay exhibited a limit of detection (LOD) of 0.01 pg/mL, and a limit of quantification of less than 0.05 pg/mL. Linearity was demonstrated across a calibration range from 0 to 100 pg/mL, giving a five-log assay range relative to the LOD. The assay exhibited good agreement with a commercially available PSA test. PSA levels were measured in 60 post-RP specimens selected as being below the detection limit of a commercially available chemiluminescent immunoassay. All specimens gave measurable PSA results in the SiMoA PSA assay, with results ranging down to 0.014 pg/mL (0.4 fM). Conclusion: The results suggest that PSA can be reliably measured in post RP patients with SiMoA technology. Recent results by others suggest measurement of nadir PSA following surgery may have prognostic value for recurrence-free survival. Measurement of PSA in post RP patients with AccuPSA could affirm a good prognosis, reduce unnecessary adjuvant radiation, and enable earlier detection of recurrence for earlier, more effective treatment.

Plasmonic Electricity: A Digital form of Metal-Enhanced Fluorescence

Fluorescence technologies are entrenched in the biosciences today. In nearly all aspects of fluorescence spectroscopy light is focused and collected by a detector which converts the photon flux into a digital signal which is then displayed. To boost optical signatures many groups have shown that the close proximity of fluorescent species to fluorophores, significantly amplifies the fluorescence signatures many fold, a technology recently described as Metal-Enhanced Fluorescence by the Geddes labs1 However, hidden within these close-range near field fluorophore-metal interactions is an induced plasmonic current, directly proportional to the excitation irradiance and the concentration of the fluorophores present in the near-field, < 20 nm. The current can be read directly, opening up huge opportunities for both the amplification and the direct detection of fluorescence, i.e. digital fluorescence, such as in solar energy conversion, digital immunoassays (Figure 1), DNA detection and in fluorescence microscopy. The direct measurement of fluorescence is likely to find profound applications and implications in the biosciences and promises to change both the way we think and use fluorescence spectroscopy today.Figure 1Metal-Enhanced Fluorescence-based digital immunoassays. A model IgG-Anti-IgG assay, demonstrating the direct detection of Fluorescence.View Large Image | View Hi-Res Image | Download PowerPoint Slide1. Metal-Enhanced Fluorescence, edited by Chris D. Geddes, John Wiley and Sons, New Jersey, 2009. - In Press.