Sensitivity and binding kinetics of an ultra-sensitive chemiluminescent enzyme-linked immunosorbent assay at arrays of antibodies

Tobos, Carmen; Kim, Seunghyeon; Rissin, David M.; Johnson, Joseph M.; Douglas, Scott L.; Yan, Susan; Nie, Shuai; Rice, B. W.; Sung, Ki-Joo; Sikes, Hadley D.; Duffy, David C.

doi:10.1016/j.jim.2019.112643

“…Indeed, Simoa ® technology can sense protein markers with attomolar (aM) LODs [ 12 , 13 ], corresponding to 10 2 −10 3 proteins in a sampled volume of 100 μL, being far less sensitive than NGS. Recently, Simoa Planar Array technology (SP-X System) [ 14 ] was developed based on a multiplex enzyme-linked immunosorbent assay (ELISA) system encompassing digital chemiluminescent imaging of an array of capture antibodies [ 15 , 16 ]. Simoa SP-X technology can sense protein markers with LODs in the sub-femtomolar range.…”

Section: Introductionmentioning

confidence: 99%

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

Sarcina

¹

,

Viola

²

,

Modena

³

et al. 2022

View full text Add to dashboard Cite

Early diagnosis in a premalignant (or pre-invasive) state represents the only chance for cure in neoplastic diseases such as pancreatic-biliary cancer, which are otherwise detected at later stages and can only be treated using palliative approaches, with no hope for a cure. Screening methods for the purpose of secondary prevention are not yet available for these cancers. Current diagnostic methods mostly rely on imaging techniques and conventional cytopathology, but they do not display adequate sensitivity to allow valid early diagnosis. Next-generation sequencing can be used to detect DNA markers down to the physical limit; however, this assay requires labeling and is time-consuming. The additional determination of a protein marker that is a predictor of aggressive behavior is a promising innovative approach, which holds the potential to improve diagnostic accuracy. Moreover, the possibility to detect biomarkers in blood serum offers the advantage of a noninvasive diagnosis. In this study, both the DNA and protein markers of pancreatic mucinous cysts were analyzed in human blood serum down to the single-molecule limit using the SiMoT (single-molecule assay with a large transistor) platform. The SiMoT device proposed herein, which exploits an inkjet-printed organic semiconductor on plastic foil, comprises an innovative 3D-printed sensing gate module, consisting of a truncated cone that protrudes from a plastic substrate and is compatible with standard ELISA wells. This 3D gate concept adds tremendous control over the biosensing system stability, along with minimal consumption of the capturing molecules and body fluid samples. The 3D sensing gate modules were extensively characterized from both a material and electrical perspective, successfully proving their suitability as detection interfaces for biosensing applications. KRAS and MUC1 target molecules were successfully analyzed in diluted human blood serum with the 3D sensing gate functionalized with b-KRAS and anti-MUC1, achieving a limit of detection of 10 zM and 40 zM, respectively. These limits of detection correspond to (1 ± 1) KRAS and (2 ± 1) MUC1 molecules in the 100 μL serum sample volume. This study provides a promising application of the 3D SiMoT platform, potentially facilitating the timely, noninvasive, and reliable identification of pancreatic cancer precursor cysts. Graphical abstract

show abstract

“…Chemie little explored primarily due to diffusion limitations. [37] Here, we inkjet-printed arrays consisting of three concentric rings of spots,e ach ring containing 9s pots with 3r eplicates for each tested IgG,for a3-plex. Thespatial distribution of each ring of spots coincides with one of the 3m esas.T he first circle of spots is located closer to the inlet mesa radius (at R = 0.33 mm) to leverage the locally higher shear rate (see SI-S6.3 for information on spot configuration).…”

Section: Methodsmentioning

confidence: 99%

Advection‐Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities

Pereiro

¹

,

Khartchenko

²

,

Lovchik

³

et al. 2021

View full text Add to dashboard Cite

Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics. However, the disruptive nature of microfluidics with existing labware and protocols has narrowed its transformative potential. We present WellProbe, a novel microfluidic concept compatible with MTPs. With it, we show that immunoassays become more sensitive at low concentrations (up to 9× signal improvement in 12× less time), richer in information with 3–4 different kinetic conditions, and can be used to estimate kinetic parameters, minimize washing steps and non‐specific binding, and identify compromised results. We further multiplex single‐well assays combining WellProbe's kinetic regions with tailored microarrays. Finally, we demonstrate our system in a context of immunoglobulin subclass evaluation, increasingly regarded as clinically relevant.

show abstract

“…Chemie Forschungsartikel little explored primarily due to diffusion limitations. [37] Here, we inkjet-printed arrays consisting of three concentric rings of spots,e ach ring containing 9s pots with 3r eplicates for each tested IgG,for a3-plex. Thespatial distribution of each ring of spots coincides with one of the 3m esas.T he first circle of spots is located closer to the inlet mesa radius (at R = 0.33 mm) to leverage the locally higher shear rate (see SI-S6.3 for information on spot configuration).…”

Section: Methodsmentioning

confidence: 99%

Advection‐Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities

Pereiro

¹

,

Khartchenko

²

,

Lovchik

³

et al. 2021

View full text Add to dashboard Cite

Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics. However, the disruptive nature of microfluidics with existing labware and protocols has narrowed its transformative potential. We present WellProbe, a novel microfluidic concept compatible with MTPs. With it, we show that immunoassays become more sensitive at low concentrations (up to 9× signal improvement in 12× less time), richer in information with 3–4 different kinetic conditions, and can be used to estimate kinetic parameters, minimize washing steps and non‐specific binding, and identify compromised results. We further multiplex single‐well assays combining WellProbe's kinetic regions with tailored microarrays. Finally, we demonstrate our system in a context of immunoglobulin subclass evaluation, increasingly regarded as clinically relevant.

show abstract

Sensitivity and binding kinetics of an ultra-sensitive chemiluminescent enzyme-linked immunosorbent assay at arrays of antibodies

Cited by 20 publications

References 30 publications

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

Advection‐Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities

Advection‐Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities

Contact Info

Product

Resources

About