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1.
Seymour, Elif; Ünlü, M. Selim; Connor, John H
A high-throughput single-particle imaging platform for antibody characterization and a novel competition assay for therapeutic antibodies Journal Article
In: Scientific Reports, vol. 13, iss. 1, pp. 1, 2023.
Abstract | Links | BibTeX | Tags:
@article{nokeyb,
title = {A high-throughput single-particle imaging platform for antibody characterization and a novel competition assay for therapeutic antibodies},
author = {Elif Seymour and M. Selim Ünlü and John H Connor},
url = {https://www.researchsquare.com/article/rs-1898923/v1},
doi = {doi.org/10.21203/rs.3.rs-1898923/v1},
year = {2023},
date = {2023-01-16},
urldate = {2022-08-03},
journal = {Scientific Reports},
volume = {13},
issue = {1},
pages = {1},
abstract = {Monoclonal antibodies (mAbs) play an important role in diagnostics and therapy of infectious diseases.
Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30
mAbs against Ebola and Lassa viruses (EBOV and LASV) to find out the ideal capture antibodies for
whole virus detection using recombinant VSV (rVSV) models expressing surface glycoproteins of EBOV
and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the
antibody epitopes on the glycoprotein structure. mAbs that bind to mucin-like domain or glycan cap of
the EBOV surface glycoprotein show the highest signal on SP-IRIS, followed by mAbs that target the GP1-
GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV
infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best
performance on SP-IRIS. This antibody binds to a unique region on the surface glycoprotein compared to
other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and
rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb
cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher
efficacy compared to other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do
not compete with each other for binding to EBOV GP. In fact, binding of 13C6 enhances the binding of
2G4 and 4G7 antibodies. SP-IRIS is a versatile tool that can provide high-throughput screening of mAbs,
multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Monoclonal antibodies (mAbs) play an important role in diagnostics and therapy of infectious diseases.
Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30
mAbs against Ebola and Lassa viruses (EBOV and LASV) to find out the ideal capture antibodies for
whole virus detection using recombinant VSV (rVSV) models expressing surface glycoproteins of EBOV
and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the
antibody epitopes on the glycoprotein structure. mAbs that bind to mucin-like domain or glycan cap of
the EBOV surface glycoprotein show the highest signal on SP-IRIS, followed by mAbs that target the GP1-
GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV
infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best
performance on SP-IRIS. This antibody binds to a unique region on the surface glycoprotein compared to
other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and
rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb
cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher
efficacy compared to other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do
not compete with each other for binding to EBOV GP. In fact, binding of 13C6 enhances the binding of
2G4 and 4G7 antibodies. SP-IRIS is a versatile tool that can provide high-throughput screening of mAbs,
multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails
Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30
mAbs against Ebola and Lassa viruses (EBOV and LASV) to find out the ideal capture antibodies for
whole virus detection using recombinant VSV (rVSV) models expressing surface glycoproteins of EBOV
and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the
antibody epitopes on the glycoprotein structure. mAbs that bind to mucin-like domain or glycan cap of
the EBOV surface glycoprotein show the highest signal on SP-IRIS, followed by mAbs that target the GP1-
GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV
infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best
performance on SP-IRIS. This antibody binds to a unique region on the surface glycoprotein compared to
other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and
rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb
cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher
efficacy compared to other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do
not compete with each other for binding to EBOV GP. In fact, binding of 13C6 enhances the binding of
2G4 and 4G7 antibodies. SP-IRIS is a versatile tool that can provide high-throughput screening of mAbs,
multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails
2.
Celebi, Iris; Aslan, Mete; Ünlü, M. Selim
A spatially uniform illumination source for widefield multi-spectral optical microscopy Journal Article Forthcoming
In: Physics Optics, Forthcoming.
Abstract | Links | BibTeX | Tags:
@article{Celebi2022,
title = {A spatially uniform illumination source for widefield multi-spectral optical microscopy},
author = {Iris Celebi and Mete Aslan and M. Selim Ünlü},
url = {https://arxiv.org/abs/2212.06216
},
doi = {https://doi.org/10.48550/arXiv.2212.06216},
year = {2022},
date = {2022-12-12},
journal = {Physics Optics},
abstract = {llumination uniformity is a critical parameter for excitation and data extraction quality in widefield biological imaging applications. However, typical imaging systems suffer from spatial and spectral non-uniformity due to non-ideal optical elements, thus require complex solutions for illumination corrections. We present Effective Uniform Color-Light Integration Device (EUCLID), a simple and cost-effective illumination source for uniformity corrections. EUCLID employs a diffuse-reflective, adjustable hollow cavity that allows for uniform mixing of light from discrete light sources and modifies the source field distribution to compensate for spatial non-uniformity introduced by optical components in the imaging system. In this study, we characterize the light coupling efficiency of the proposed design and compare the uniformity performance with the conventional method. EUCLID demonstrates a remarkable illumination improvement for multi-spectral imaging in both Nelsonian and Koehler alignment with a maximum spatial deviation of ~1% across a wide field-of-view.},
keywords = {},
pubstate = {forthcoming},
tppubtype = {article}
}
llumination uniformity is a critical parameter for excitation and data extraction quality in widefield biological imaging applications. However, typical imaging systems suffer from spatial and spectral non-uniformity due to non-ideal optical elements, thus require complex solutions for illumination corrections. We present Effective Uniform Color-Light Integration Device (EUCLID), a simple and cost-effective illumination source for uniformity corrections. EUCLID employs a diffuse-reflective, adjustable hollow cavity that allows for uniform mixing of light from discrete light sources and modifies the source field distribution to compensate for spatial non-uniformity introduced by optical components in the imaging system. In this study, we characterize the light coupling efficiency of the proposed design and compare the uniformity performance with the conventional method. EUCLID demonstrates a remarkable illumination improvement for multi-spectral imaging in both Nelsonian and Koehler alignment with a maximum spatial deviation of ~1% across a wide field-of-view.
3.
Kanik, Fulya Ekiz; Celebi, Iris; Sevenler, Derin; Tanriverdi, Kahraman; Ünlü, Nese Lortlar; Freedman, Jane E.; Ünlü, M. Selim
Attomolar sensitivity microRNA detection using real‑time digital microarrays Journal Article
In: Scientific Reports, vol. 2022, iss. 2, no. 12, pp. 16220, 2022.
Abstract | Links | BibTeX | Tags: Oligo
@article{nokey,
title = {Attomolar sensitivity microRNA detection using real‑time digital microarrays},
author = {Fulya Ekiz Kanik and Iris Celebi and Derin Sevenler and Kahraman Tanriverdi and Nese Lortlar Ünlü and Jane E. Freedman and M. Selim Ünlü},
url = {https://mhg912.p3cdn1.secureserver.net/wp-content/uploads/2022/09/Attomolar-sensitivity-microRNA-detection.pdf},
doi = {10.1038/s41598-022-19912-z},
year = {2022},
date = {2022-09-27},
urldate = {2022-09-27},
booktitle = {Imaging Systems and Applications},
journal = {Scientific Reports},
volume = {2022},
number = {12},
issue = {2},
pages = {16220},
publisher = {Optica Publishing Group},
series = {Biophysics At the Nanoscale},
abstract = {MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA‑451a (miR‑451) and miRNA‑223‑3p (miR‑223). We demonstrated improvements in sensitivity in comparison to traditional end‑point assays that employ capture on solid phase support, by implementing real‑time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low‑abundance biomarkers in the presence
of low‑affinity but high‑abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to ∼ 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.},
howpublished = {https://chemrxiv.org/engage/chemrxiv/article-details/6565fc1829a13c4d4730f055},
keywords = {Oligo},
pubstate = {published},
tppubtype = {article}
}
MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA‑451a (miR‑451) and miRNA‑223‑3p (miR‑223). We demonstrated improvements in sensitivity in comparison to traditional end‑point assays that employ capture on solid phase support, by implementing real‑time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low‑abundance biomarkers in the presence
of low‑affinity but high‑abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to ∼ 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.
of low‑affinity but high‑abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to ∼ 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.
4.
Kanik, Fulya Ekiz; Celebi, Iris; Sevenler, Derin; Tanriverdi, Kahraman; Ünlü, Nese Lortlar; Freedman, Jane E.; Ünlü, M. Selim
Attomolar sensitivity microRNA detection using real‑time digital microarrays Journal Article
In: Scientific Reports, vol. 2022, no. 12, pp. 16220, 2022.
Abstract | Links | BibTeX | Tags: Oligo
@article{nokey,
title = {Attomolar sensitivity microRNA detection using real‑time digital microarrays},
author = {Fulya Ekiz Kanik and Iris Celebi and Derin Sevenler and Kahraman Tanriverdi and Nese Lortlar Ünlü and Jane E. Freedman and M. Selim Ünlü},
url = {https://mhg912.p3cdn1.secureserver.net/wp-content/uploads/2022/09/Attomolar-sensitivity-microRNA-detection.pdf},
doi = {10.1038/s41598-022-19912-z},
year = {2022},
date = {2022-09-27},
urldate = {2022-09-27},
journal = {Scientific Reports},
volume = {2022},
number = {12},
pages = {16220},
abstract = {MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA‑451a (miR‑451) and miRNA‑223‑3p (miR‑223). We demonstrated improvements in sensitivity in comparison to traditional end‑point assays that employ capture on solid phase support, by implementing real‑time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low‑abundance biomarkers in the presence
of low‑affinity but high‑abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to ∼ 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.},
keywords = {Oligo},
pubstate = {published},
tppubtype = {article}
}
MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA‑451a (miR‑451) and miRNA‑223‑3p (miR‑223). We demonstrated improvements in sensitivity in comparison to traditional end‑point assays that employ capture on solid phase support, by implementing real‑time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low‑abundance biomarkers in the presence
of low‑affinity but high‑abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to ∼ 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.
of low‑affinity but high‑abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to ∼ 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.
5.
Seymour, Elif; Ünlü, M. Selim; Connor, John H
A high-throughput single-particle imaging platform for antibody characterization and a novel competition assay for therapeutic antibodies Journal Article Forthcoming
In: https://www.researchsquare.com/article/rs-1898923/v1, Forthcoming.
Abstract | Links | BibTeX | Tags:
@article{nokey,
title = {A high-throughput single-particle imaging platform for antibody characterization and a novel competition assay for therapeutic antibodies},
author = {Elif Seymour and M. Selim Ünlü and John H Connor},
url = {https://www.researchsquare.com/article/rs-1898923/v1
},
doi = {doi.org/10.21203/rs.3.rs-1898923/v1},
year = {2022},
date = {2022-08-03},
urldate = {2022-08-03},
journal = {https://www.researchsquare.com/article/rs-1898923/v1},
abstract = {Monoclonal antibodies (mAbs) play an important role in diagnostics and therapy of infectious diseases.
Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30
mAbs against Ebola and Lassa viruses (EBOV and LASV) to find out the ideal capture antibodies for
whole virus detection using recombinant VSV (rVSV) models expressing surface glycoproteins of EBOV
and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the
antibody epitopes on the glycoprotein structure. mAbs that bind to mucin-like domain or glycan cap of
the EBOV surface glycoprotein show the highest signal on SP-IRIS, followed by mAbs that target the GP1-
GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV
infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best
performance on SP-IRIS. This antibody binds to a unique region on the surface glycoprotein compared to
other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and
rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb
cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher
efficacy compared to other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do
not compete with each other for binding to EBOV GP. In fact, binding of 13C6 enhances the binding of
2G4 and 4G7 antibodies. SP-IRIS is a versatile tool that can provide high-throughput screening of mAbs,
multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails},
keywords = {},
pubstate = {forthcoming},
tppubtype = {article}
}
Monoclonal antibodies (mAbs) play an important role in diagnostics and therapy of infectious diseases.
Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30
mAbs against Ebola and Lassa viruses (EBOV and LASV) to find out the ideal capture antibodies for
whole virus detection using recombinant VSV (rVSV) models expressing surface glycoproteins of EBOV
and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the
antibody epitopes on the glycoprotein structure. mAbs that bind to mucin-like domain or glycan cap of
the EBOV surface glycoprotein show the highest signal on SP-IRIS, followed by mAbs that target the GP1-
GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV
infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best
performance on SP-IRIS. This antibody binds to a unique region on the surface glycoprotein compared to
other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and
rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb
cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher
efficacy compared to other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do
not compete with each other for binding to EBOV GP. In fact, binding of 13C6 enhances the binding of
2G4 and 4G7 antibodies. SP-IRIS is a versatile tool that can provide high-throughput screening of mAbs,
multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails
Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30
mAbs against Ebola and Lassa viruses (EBOV and LASV) to find out the ideal capture antibodies for
whole virus detection using recombinant VSV (rVSV) models expressing surface glycoproteins of EBOV
and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the
antibody epitopes on the glycoprotein structure. mAbs that bind to mucin-like domain or glycan cap of
the EBOV surface glycoprotein show the highest signal on SP-IRIS, followed by mAbs that target the GP1-
GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV
infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best
performance on SP-IRIS. This antibody binds to a unique region on the surface glycoprotein compared to
other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and
rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb
cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher
efficacy compared to other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do
not compete with each other for binding to EBOV GP. In fact, binding of 13C6 enhances the binding of
2G4 and 4G7 antibodies. SP-IRIS is a versatile tool that can provide high-throughput screening of mAbs,
multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails
6.
Bakhshpour, M; Chiodi, E; Celebi, I; Saylan, Y; Ünlü, NL; Ünlü, MS; Denizli, A
Sensitive and real-time detection of IgG using interferometric reflecting imaging sensor system Journal Article
In: Biosensors and Bioelectronics, vol. 201, pp. 113961, 2022.
Abstract | Links | BibTeX | Tags: Protein
@article{nokeyc,
title = {Sensitive and real-time detection of IgG using interferometric reflecting imaging sensor system},
author = {M Bakhshpour and E Chiodi and I Celebi and Y Saylan and NL Ünlü and MS Ünlü and A Denizli},
doi = {https://doi.org/10.1016/j.bios.2021.113961},
year = {2022},
date = {2022-04-01},
urldate = {2022-04-01},
journal = {Biosensors and Bioelectronics},
volume = {201},
pages = {113961},
abstract = {Considering the limitations of well-known traditional detection techniques, innovative research studies have focused on the development of new sensors to offer label-free, highly sensitive, real-time, low-cost, and rapid detection for biomolecular interactions. In this study, we demonstrate immunoglobulin G (IgG) detection in aqueous solutions by using real-time and label-free kinetic measurements of the Interferometric Reflectance Imaging Sensor (IRIS) system. By performing kinetic characterization experiments, the sensor's performance is comprehensively evaluated and a high correlation coefficient value (>0.94) is obtained in the IgG concentration range of 1–50 μg/mL with a low detection limit (0.25 μg/mL or 1.67 nM). Moreover, the highly sensitive imaging system ensures accurate quantification and reliable validation of recorded binding events, offering new perspectives in terms of direct biomarker detection for clinical applications.},
keywords = {Protein},
pubstate = {published},
tppubtype = {article}
}
Considering the limitations of well-known traditional detection techniques, innovative research studies have focused on the development of new sensors to offer label-free, highly sensitive, real-time, low-cost, and rapid detection for biomolecular interactions. In this study, we demonstrate immunoglobulin G (IgG) detection in aqueous solutions by using real-time and label-free kinetic measurements of the Interferometric Reflectance Imaging Sensor (IRIS) system. By performing kinetic characterization experiments, the sensor's performance is comprehensively evaluated and a high correlation coefficient value (>0.94) is obtained in the IgG concentration range of 1–50 μg/mL with a low detection limit (0.25 μg/mL or 1.67 nM). Moreover, the highly sensitive imaging system ensures accurate quantification and reliable validation of recorded binding events, offering new perspectives in terms of direct biomarker detection for clinical applications.
7.
Chiodi, E; Marn, AM; Bakhshpour, M; ans MS Ünlü, N Lortlar Ünlü
The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors Journal Article
In: Polymers, vol. 14, iss. 2, pp. 241, 2022.
Abstract | Links | BibTeX | Tags: Oligo, Protein
@article{nokey,
title = {The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors},
author = {E Chiodi and AM Marn and M Bakhshpour and N Lortlar Ünlü ans MS Ünlü},
editor = {Ick-Soo Kim},
url = {https://www.mdpi.com/2073-4360/14/2/241},
doi = {https://doi.org/10.3390/polym14020241},
year = {2022},
date = {2022-01-07},
urldate = {2022-01-07},
journal = {Polymers},
volume = {14},
issue = {2},
pages = {241},
abstract = {The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches},
keywords = {Oligo, Protein},
pubstate = {published},
tppubtype = {article}
}
The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches
8.
Chiodi, E; Marn, AM; Bakhshpour, M; Ünlü, N Lortlar Ünlü MS
The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors Journal Article
In: Polymers, vol. 14, iss. 2, pp. 241, 2022.
Abstract | Links | BibTeX | Tags: Oligo, Protein
@article{nokeyd,
title = {The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors},
author = {E Chiodi and AM Marn and M Bakhshpour and N Lortlar Ünlü MS Ünlü},
editor = {Ick-Soo Kim},
url = {https://www.mdpi.com/2073-4360/14/2/241},
doi = {https://doi.org/10.3390/polym14020241},
year = {2022},
date = {2022-01-07},
urldate = {2022-01-07},
journal = {Polymers},
volume = {14},
issue = {2},
pages = {241},
abstract = {The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches},
keywords = {Oligo, Protein},
pubstate = {published},
tppubtype = {article}
}
The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches
9.
Marn, Allison M; Needham, James; Chiodi, Elisa; Ünlü, M Selim
Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor Journal Article
In: Biosensors and Bioelectronics , vol. 11, iss. 12, pp. 483, 2021.
Abstract | Links | BibTeX | Tags: Protein
@article{nokey,
title = {Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor},
author = {Allison M Marn and James Needham and Elisa Chiodi and M Selim Ünlü},
url = {https://www.mdpi.com/2079-6374/11/12/483/htm},
doi = {https://doi.org/10.3390/bios11120483},
year = {2021},
date = {2021-12-01},
urldate = {2021-12-01},
journal = {Biosensors and Bioelectronics },
volume = {11},
issue = {12},
pages = {483},
abstract = {Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.},
keywords = {Protein},
pubstate = {published},
tppubtype = {article}
}
Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.
10.
Marn, Allison M; Needham, James; Chiodi, Elisa; Ünlü, M Selim
Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor Journal Article
In: Biosensors and Bioelectronics, vol. 11, iss. 12, pp. 483, 2021.
Abstract | Links | BibTeX | Tags: Protein
@article{nokeye,
title = {Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor},
author = {Allison M Marn and James Needham and Elisa Chiodi and M Selim Ünlü},
url = {https://www.mdpi.com/2079-6374/11/12/483/htm},
doi = {https://doi.org/10.3390/bios11120483},
year = {2021},
date = {2021-12-01},
urldate = {2021-12-01},
journal = {Biosensors and Bioelectronics},
volume = {11},
issue = {12},
pages = {483},
abstract = {Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.},
keywords = {Protein},
pubstate = {published},
tppubtype = {article}
}
Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.
11.
Zong, Haonan; Yurdakul, Celalettin; Bai, Yeran; Zhang, Meng; Ünlü, M. Selim; Cheng, Ji-Xin
Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering Journal Article
In: ACS Photonics Article ASAP, vol. 8, iss. 11, pp. 3323, 2021.
Abstract | Links | BibTeX | Tags: Bacteria
@article{nokey,
title = {Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering},
author = {Haonan Zong and Celalettin Yurdakul and Yeran Bai and Meng Zhang and M. Selim Ünlü and Ji-Xin Cheng},
url = {https://arxiv.org/pdf/2104.06247},
doi = {https://doi.org/10.1021/acsphotonics.1c01197},
year = {2021},
date = {2021-10-14},
urldate = {2021-10-14},
journal = {ACS Photonics Article ASAP},
volume = {8},
issue = {11},
pages = {3323},
abstract = {Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from subwavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over 3 orders of magnitude background suppression by quasi-darkfield illumination in the epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 μm × 70 μm. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500 nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria and MIP imaging of subcellular lipid droplets inside C. albicans and cancer cells.},
keywords = {Bacteria},
pubstate = {published},
tppubtype = {article}
}
Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from subwavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over 3 orders of magnitude background suppression by quasi-darkfield illumination in the epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 μm × 70 μm. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500 nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria and MIP imaging of subcellular lipid droplets inside C. albicans and cancer cells.
12.
Zong, Haonan; Yurdakul, Celalettin; Bai, Yeran; Zhang, Meng; Ünlü, M. Selim; Cheng, Ji-Xin
Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering Journal Article
In: ACS Photonics Article ASAP, vol. 8, iss. 11, pp. 3323, 2021.
Abstract | Links | BibTeX | Tags: Bacteria
@article{nokeyf,
title = {Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering},
author = {Haonan Zong and Celalettin Yurdakul and Yeran Bai and Meng Zhang and M. Selim Ünlü and Ji-Xin Cheng},
url = {https://arxiv.org/pdf/2104.06247},
doi = {https://doi.org/10.1021/acsphotonics.1c01197},
year = {2021},
date = {2021-10-14},
urldate = {2021-10-14},
journal = {ACS Photonics Article ASAP},
volume = {8},
issue = {11},
pages = {3323},
abstract = {Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from subwavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over 3 orders of magnitude background suppression by quasi-darkfield illumination in the epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 μm × 70 μm. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500 nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria and MIP imaging of subcellular lipid droplets inside C. albicans and cancer cells.},
keywords = {Bacteria},
pubstate = {published},
tppubtype = {article}
}
Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from subwavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over 3 orders of magnitude background suppression by quasi-darkfield illumination in the epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 μm × 70 μm. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500 nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria and MIP imaging of subcellular lipid droplets inside C. albicans and cancer cells.
13.
Priest,; Peters, Jack S.; Kukura, Philipp
Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins Journal Article
In: Chem. Rev., vol. 121, iss. 19, pp. 11937-11970, 2021.
Abstract | Links | BibTeX | Tags:
@article{nokeyg,
title = {Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins},
author = {Priest and Jack S. Peters and Philipp Kukura},
url = {https://pubs.acs.org/doi/full/10.1021/acs.chemrev.1c00271},
doi = {https://doi.org/10.1021/acs.chemrev.1c00271},
year = {2021},
date = {2021-09-29},
journal = {Chem. Rev.},
volume = {121},
issue = {19},
pages = {11937-11970},
abstract = {Our ability to detect, image, and quantify nanoscopic objects and molecules with visible light has undergone dramatic improvements over the past few decades. While fluorescence has historically been the go-to contrast mechanism for ultrasensitive light microscopy due to its superior background suppression and specificity, recent developments based on light scattering have reached single-molecule sensitivity. They also have the advantages of universal applicability and the ability to obtain information about the species of interest beyond its presence and location. Many of the recent advances are driven by novel approaches to illumination, detection, and background suppression, all aimed at isolating and maximizing the signal of interest. Here, we review these developments grouped according to the basic principles used, namely darkfield imaging, interferometric detection, and surface plasmon resonance microscopy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Our ability to detect, image, and quantify nanoscopic objects and molecules with visible light has undergone dramatic improvements over the past few decades. While fluorescence has historically been the go-to contrast mechanism for ultrasensitive light microscopy due to its superior background suppression and specificity, recent developments based on light scattering have reached single-molecule sensitivity. They also have the advantages of universal applicability and the ability to obtain information about the species of interest beyond its presence and location. Many of the recent advances are driven by novel approaches to illumination, detection, and background suppression, all aimed at isolating and maximizing the signal of interest. Here, we review these developments grouped according to the basic principles used, namely darkfield imaging, interferometric detection, and surface plasmon resonance microscopy.
14.
lee Priest,; Peters, Jack S.; Kukura, Philipp
Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins Journal Article
In: Chem. Rev., vol. 121, iss. 19, pp. 11937-11970, 2021.
Abstract | Links | BibTeX | Tags:
@article{nokey,
title = {Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins},
author = {lee Priest and Jack S. Peters and Philipp Kukura},
url = {https://pubs.acs.org/doi/full/10.1021/acs.chemrev.1c00271},
doi = {https://doi.org/10.1021/acs.chemrev.1c00271},
year = {2021},
date = {2021-09-29},
journal = {Chem. Rev.},
volume = {121},
issue = {19},
pages = {11937-11970},
abstract = {Our ability to detect, image, and quantify nanoscopic objects and molecules with visible light has undergone dramatic improvements over the past few decades. While fluorescence has historically been the go-to contrast mechanism for ultrasensitive light microscopy due to its superior background suppression and specificity, recent developments based on light scattering have reached single-molecule sensitivity. They also have the advantages of universal applicability and the ability to obtain information about the species of interest beyond its presence and location. Many of the recent advances are driven by novel approaches to illumination, detection, and background suppression, all aimed at isolating and maximizing the signal of interest. Here, we review these developments grouped according to the basic principles used, namely darkfield imaging, interferometric detection, and surface plasmon resonance microscopy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Our ability to detect, image, and quantify nanoscopic objects and molecules with visible light has undergone dramatic improvements over the past few decades. While fluorescence has historically been the go-to contrast mechanism for ultrasensitive light microscopy due to its superior background suppression and specificity, recent developments based on light scattering have reached single-molecule sensitivity. They also have the advantages of universal applicability and the ability to obtain information about the species of interest beyond its presence and location. Many of the recent advances are driven by novel approaches to illumination, detection, and background suppression, all aimed at isolating and maximizing the signal of interest. Here, we review these developments grouped according to the basic principles used, namely darkfield imaging, interferometric detection, and surface plasmon resonance microscopy.
15.
Yurdakul, Celalettin
2021.
Abstract | Links | BibTeX | Tags:
@phdthesis{nokeyh,
title = {Interferometric Reflectance Microscopy for Physical and Chemical Characterization of Biological Nanoparticles},
author = {Celalettin Yurdakul},
url = {https://www.proquest.com/openview/68b695b3f62a6733151e588aacb3b868/1?pq-origsite=gscholar&cbl=18750&diss=y},
year = {2021},
date = {2021-07-01},
abstract = {Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles.
The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system’s classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts.},
howpublished = {Boston University Dissertation},
keywords = {},
pubstate = {published},
tppubtype = {phdthesis}
}
Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles.
The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system’s classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts.
The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system’s classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts.
16.
Yurdakul, Celalettin
2021.
Abstract | Links | BibTeX | Tags:
@phdthesis{nokey,
title = {Interferometric Reflectance Microscopy for Physical and Chemical Characterization of Biological Nanoparticles},
author = {Celalettin Yurdakul},
url = {https://www.proquest.com/openview/68b695b3f62a6733151e588aacb3b868/1?pq-origsite=gscholar&cbl=18750&diss=y},
year = {2021},
date = {2021-07-01},
abstract = {Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles.
The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system’s classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts.},
howpublished = {Boston University Dissertation},
keywords = {},
pubstate = {published},
tppubtype = {phdthesis}
}
Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles.
The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system’s classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts.
The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system’s classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts.
17.
Yurdakul, Celalettin; Zong, Haonan; Bai, Yeran; Cheng, Ji-Xin; Ünlü, M Selim
Bond-selective interferometric scattering microscopy Journal Article
In: Journal of Physics D: Applied Physics, vol. 54, iss. 36, pp. 364002, 2021.
Abstract | Links | BibTeX | Tags: Bacteria
@article{nokey,
title = {Bond-selective interferometric scattering microscopy},
author = {Celalettin Yurdakul and Haonan Zong and Yeran Bai and Ji-Xin Cheng and M Selim Ünlü},
url = {https://arxiv.org/pdf/2106.02931},
doi = {https://doi.org/10.1088/1361-6463/ac0b0d},
year = {2021},
date = {2021-06-25},
urldate = {2021-06-25},
journal = {Journal of Physics D: Applied Physics},
volume = {54},
issue = {36},
pages = {364002},
abstract = {Interferometric scattering (iSCAT) microscopy has been a very promising technology for highly sensitive label-free imaging of a broad spectrum of biological nanoparticles from proteins to viruses in a high-throughput manner. Although it can reveal the specimen's size and shape information, the chemical composition is inaccessible in interferometric measurements. Infrared (IR) spectroscopic imaging provides chemical specificity based on inherent chemical bond vibrations of specimens but lacks the ability to image and resolve individual nanoparticles due to long IR wavelengths. Here, we describe a bond-selective iSCAT microscope where the mid-IR induced photothermal signal is detected by a visible beam in a wide-field common-path interferometry configuration. A thin film layered substrate is utilized to reduce the reflected light and provide a reference field for the interferometric detection of the weakly scattered field. A pulsed mid-IR laser is employed to modulate the interferometric signal. Subsequent demodulation via a virtual lock-in camera offers simultaneous chemical information about tens of micro- or nano-particles. The chemical contrast arises from a minute change in the particle's scattered field in consequence of the vibrational absorption at the target molecule. We characterize the system with sub-wavelength polymer beads and highlight biological applications by chemically imaging several microorganisms including Staphylococcus aureus, Escherichia coli, and Candida albicans. A theoretical framework is established to extend bond-selective iSCAT microscopy to a broad range of biological micro- and nano-particles.},
keywords = {Bacteria},
pubstate = {published},
tppubtype = {article}
}
Interferometric scattering (iSCAT) microscopy has been a very promising technology for highly sensitive label-free imaging of a broad spectrum of biological nanoparticles from proteins to viruses in a high-throughput manner. Although it can reveal the specimen's size and shape information, the chemical composition is inaccessible in interferometric measurements. Infrared (IR) spectroscopic imaging provides chemical specificity based on inherent chemical bond vibrations of specimens but lacks the ability to image and resolve individual nanoparticles due to long IR wavelengths. Here, we describe a bond-selective iSCAT microscope where the mid-IR induced photothermal signal is detected by a visible beam in a wide-field common-path interferometry configuration. A thin film layered substrate is utilized to reduce the reflected light and provide a reference field for the interferometric detection of the weakly scattered field. A pulsed mid-IR laser is employed to modulate the interferometric signal. Subsequent demodulation via a virtual lock-in camera offers simultaneous chemical information about tens of micro- or nano-particles. The chemical contrast arises from a minute change in the particle's scattered field in consequence of the vibrational absorption at the target molecule. We characterize the system with sub-wavelength polymer beads and highlight biological applications by chemically imaging several microorganisms including Staphylococcus aureus, Escherichia coli, and Candida albicans. A theoretical framework is established to extend bond-selective iSCAT microscopy to a broad range of biological micro- and nano-particles.
18.
Yurdakul, Celalettin; Zong, Haonan; Bai, Yeran; Cheng, Ji-Xin; Ünlü, M Selim
Bond-selective interferometric scattering microscopy Journal Article
In: Journal of Physics D: Applied Physics, vol. 54, iss. 36, pp. 364002, 2021.
Abstract | Links | BibTeX | Tags: Bacteria
@article{nokeyi,
title = {Bond-selective interferometric scattering microscopy},
author = {Celalettin Yurdakul and Haonan Zong and Yeran Bai and Ji-Xin Cheng and M Selim Ünlü},
url = {https://arxiv.org/pdf/2106.02931},
doi = {https://doi.org/10.1088/1361-6463/ac0b0d},
year = {2021},
date = {2021-06-25},
urldate = {2021-06-25},
journal = {Journal of Physics D: Applied Physics},
volume = {54},
issue = {36},
pages = {364002},
abstract = {Interferometric scattering (iSCAT) microscopy has been a very promising technology for highly sensitive label-free imaging of a broad spectrum of biological nanoparticles from proteins to viruses in a high-throughput manner. Although it can reveal the specimen's size and shape information, the chemical composition is inaccessible in interferometric measurements. Infrared (IR) spectroscopic imaging provides chemical specificity based on inherent chemical bond vibrations of specimens but lacks the ability to image and resolve individual nanoparticles due to long IR wavelengths. Here, we describe a bond-selective iSCAT microscope where the mid-IR induced photothermal signal is detected by a visible beam in a wide-field common-path interferometry configuration. A thin film layered substrate is utilized to reduce the reflected light and provide a reference field for the interferometric detection of the weakly scattered field. A pulsed mid-IR laser is employed to modulate the interferometric signal. Subsequent demodulation via a virtual lock-in camera offers simultaneous chemical information about tens of micro- or nano-particles. The chemical contrast arises from a minute change in the particle's scattered field in consequence of the vibrational absorption at the target molecule. We characterize the system with sub-wavelength polymer beads and highlight biological applications by chemically imaging several microorganisms including Staphylococcus aureus, Escherichia coli, and Candida albicans. A theoretical framework is established to extend bond-selective iSCAT microscopy to a broad range of biological micro- and nano-particles.},
keywords = {Bacteria},
pubstate = {published},
tppubtype = {article}
}
Interferometric scattering (iSCAT) microscopy has been a very promising technology for highly sensitive label-free imaging of a broad spectrum of biological nanoparticles from proteins to viruses in a high-throughput manner. Although it can reveal the specimen's size and shape information, the chemical composition is inaccessible in interferometric measurements. Infrared (IR) spectroscopic imaging provides chemical specificity based on inherent chemical bond vibrations of specimens but lacks the ability to image and resolve individual nanoparticles due to long IR wavelengths. Here, we describe a bond-selective iSCAT microscope where the mid-IR induced photothermal signal is detected by a visible beam in a wide-field common-path interferometry configuration. A thin film layered substrate is utilized to reduce the reflected light and provide a reference field for the interferometric detection of the weakly scattered field. A pulsed mid-IR laser is employed to modulate the interferometric signal. Subsequent demodulation via a virtual lock-in camera offers simultaneous chemical information about tens of micro- or nano-particles. The chemical contrast arises from a minute change in the particle's scattered field in consequence of the vibrational absorption at the target molecule. We characterize the system with sub-wavelength polymer beads and highlight biological applications by chemically imaging several microorganisms including Staphylococcus aureus, Escherichia coli, and Candida albicans. A theoretical framework is established to extend bond-selective iSCAT microscopy to a broad range of biological micro- and nano-particles.
19.
Chiodi, Elisa; Daaboul, George G.; Marn, Allison M; Ünlü, M. Selim
Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics Journal Article
In: Sensors, vol. 21, iss. 8, pp. 2634, 2021.
Abstract | Links | BibTeX | Tags:
@article{nokey,
title = {Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics},
author = {Elisa Chiodi and George G. Daaboul and Allison M Marn and M. Selim Ünlü},
url = {https://www.mdpi.com/1424-8220/21/8/2634},
doi = {https://doi.org/10.3390/s21082634},
year = {2021},
date = {2021-04-09},
journal = {Sensors},
volume = {21},
issue = {8},
pages = {2634},
abstract = {Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.
20.
Chiodi, Elisa; Daaboul, George G.; Marn, Allison M; Ünlü, M. Selim
Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics Journal Article
In: Sensors, vol. 21, iss. 8, pp. 2634, 2021.
Abstract | Links | BibTeX | Tags:
@article{nokeyj,
title = {Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics},
author = {Elisa Chiodi and George G. Daaboul and Allison M Marn and M. Selim Ünlü},
url = {https://www.mdpi.com/1424-8220/21/8/2634},
doi = {https://doi.org/10.3390/s21082634},
year = {2021},
date = {2021-04-09},
journal = {Sensors},
volume = {21},
issue = {8},
pages = {2634},
abstract = {Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.
